Camptothecin Conjugates of Anti-CD22 Antibodies for Treatment of B Cell Diseases

ABSTRACT

Disclosed herein are compositions and methods of use comprising combinations of anti-CD22 antibodies with a therapeutic agent. The therapeutic agent may be attached to the anti-CD22 antibody or may be separately administered, either before, simultaneously with or after the anti-CD22 antibody. In preferred embodiments, the therapeutic agent is an antibody or fragment thereof that binds to an antigen different from CD22, such as CD 19, CD20, CD21, CD22, CD23, CD37, CD40, CD40L, CD52, CD80 and HLA-DR. However, the therapeutic agent may an immunomodulator, a cytokine, a toxin or other therapeutic agent known in the art. More preferably, the anti-CD22 antibody is part of a DNL complex, such as a hexavalent DNL complex. Most preferably, combination therapy with the anti-CD22 antibody or fragment and the therapeutic agent is more effective than the antibody alone, the therapeutic agent alone, or the combination of anti-CD22 antibody and therapeutic agent that are not conjugated to each other. Administration of the anti-CD22 antibody and therapeutic agent induces apoptosis and cell death of target cells in diseases such as B-cell lymphomas or leukemias, autoimmune disease or immune dysfunction disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/213,245, filed Aug. 19, 2011, which claims the benefit under 35U.S.C. 119(e) of provisional U.S. Patent Appl. Ser. No. 61/375,068,filed Aug. 19, 2010. U.S. patent application Ser. No. 13/213,245 acontinuation-in-part of U.S. patent application Ser. No. 13/164,275,filed Jun. 20, 2011; which was a divisional of U.S. patent applicationSer. No. 12/629,404, filed Dec. 2, 2009; which was acontinuation-in-part of U.S. patent application Ser. No. 12/026,811,filed Feb. 6, 2008; which was a continuation-in-part of U.S. patentapplication Ser. No. 11/388,032, filed Mar. 23, 2006; which was acontinuation-in-part of U.S. patent application Ser. No. 10/734,589,filed Dec. 15, 2003. Those applications claimed the benefit under 35U.S.C. 119(e) of U.S. Provisional Patent Appl. Ser. Nos. 61/207,890,filed Feb. 13, 2009; 60/751,196, filed Dec. 16, 2005; 60/728,292, filedOct. 19, 2005; 60/668,603, filed Apr. 6, 2005 and 60/433,017, filed Dec.13, 2002. The text of each priority application is incorporated hereinby reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 18, 2011, isnamed IMM317US.txt and is 59,124 bytes in size.

FIELD OF THE INVENTION

The present invention concerns compositions and methods of use ofimmunoconjugates, comprising one or more camptothecin moieties attachedto an anti-CD22 antibody or antigen-binding fragment thereof.Preferably, the anti-CD22 antibody is epratuzumab and the camptothecinis SN-38. The immunoconjugate is of use to treat B cell diseases, suchas hematologic tumors, B cell leukemia or lymphoma (e.g., mantle celllymphoma, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma,diffuse large B cell lymphoma, Burkitt lymphoma, follicular lymphoma,acute lymphocytic leukemia, chronic lymphocytic leukemia, and hairy cellleukemia), autoimmune disease, immune dysfunction disease and type 1 ortype 2 diabetes. The immunoconjugate may be used alone or may becombined with another anti-B cell antibody or fragment thereof, such asantibodies against CD 19, CD20, CD21, CD23, CD37, CD40, CD40L, CD52,CD80 or HLA-DR. Alternatively, the immunoconjugate may be used incombination with another therapeutic agent, such as an immunomodulator,a cytotoxic agent, a drug, a toxin, an anti-angiogenic agent, aproapoptotic agent or a radionuclide. The anti-CD22 immunoconjugate mayalso be administered as part of a dock-and-lock (DNL) complex, asdescribed in detail below. In most preferred embodiments, thecombination of anti-CD22 antibody and other therapeutic agent issignificantly more efficacious for treating a B cell disease than eitheragent administered alone or the sum of effects of the agentsadministered separately.

BACKGROUND

An aim of targeted drug therapy is to use monoclonal antibodies (MAbs)for the specific delivery of toxic agents to human cancers. Conjugatesof tumor-associated MAbs and suitable toxic agents have been developed,but have had mixed success in the therapy of cancer, and virtually noapplication in other diseases, such as autoimmune diseases. The toxicagent is most commonly a chemotherapeutic drug, althoughparticle-emitting radionuclides, or bacterial or plant toxins have alsobeen conjugated to MAbs, especially for the therapy of cancer (Sharkeyand Goldenberg, C A Cancer J. Clin. 2006 July-August; 56(4):226-243).

The advantages of using MAb-chemotherapeutic drug conjugates are that(a) the chemotherapeutic drug itself is structurally well defined; (b)the chemotherapeutic drug is linked to the MAb protein using very welldefined conjugation chemistries, often at specific sites remote from theMAb antigen binding regions; (c) MAb-chemotherapeutic drug conjugatescan be made more reproducibly than chemical conjugates involving MAbsand bacterial or plant toxins, and as such are more amenable tocommercial development and regulatory approval; and (d) theMAb-chemotherapeutic drug conjugates are orders of magnitude less toxicsystemically than radionuclide MAb conjugates.

Early work on protein-drug conjugates indicated that a drug preferablyis released in its original form, once it has been internalized into atarget cell, for the protein-chemotherapeutic drug conjugate to be auseful therapeutic. Trouet et al. (Proc. Natl. Acad. Sci. USA 79:626-629(1982)) showed the advantage of using specific peptide linkers, betweenthe drug and the antibody, which are cleaved lysosomally to liberate theintact drug. MAb-chemotherapeutic drug conjugates prepared using mildacid-cleavable linkers, such as those containing a hydrazone, weredeveloped, based on the observation that the pH inside tumors was oftenlower than normal physiological pH (Willner et al., U.S. Pat. No.5,708,146; Trail et al. (Science 261:212-215 (1993)). The first approvedMAb-drug conjugate, Gemtuzumab Ozogamicin, incorporated a similaracid-labile hydrazone bond between an anti-CD33 antibody, humanizedP67.6, and a potent calicheamicin derivative. Sievers et al., J ClinOncol. 19:3244-3254 (2001); Hamann et al., Bioconjugate Chem. 13: 47-58(2002). In some cases, the MAb-chemotherapeutic drug conjugates weremade with reductively labile hindered disulfide bonds between thechemotherapeutic drugs and the MAb (Liu et al., Proc Natl Acad Sci USA93: 8618-8623 (1996)).

Yet another cleavable linker involves cathepsin B-labile dipeptidespacers, such as Phe-Lys or Val-Cit, similar to the lysosomally labilepeptide spacers of Trouet et al. containing from one to four aminoacids, which additionally incorporated a collapsible spacer between thedrug and the dipeptide (Dubowchik, et al., Bioconjugate Chem. 13:855-869(2002); Firestone et al., U.S. Pat. No. 6,214,345 B 1; Doronina et al.,Nat. Biotechnol. 21: 778-784 (2003)). The latter approaches were alsoutilized in the preparation of an immunoconjugate of camptothecin(Walker et al., Bioorg Med Chem. Lett. 12:217-219 (2002)). Anothercleavable moiety that has been explored is an ester linkage incorporatedinto the linker between the antibody and the chemotherapeutic drug.Gillimard and Saragovi have found that when an ester of paclitaxel wasconjugated to anti-rat p75 MAb, MC192, or anti-human TrkA MAb, 5C3, theconjugate was found to exhibit target-specific toxicity. Gillimard andSaragovi, Cancer Res. 61:694-699 (2001).

The conjugates of the instant invention possess greater efficacy, inmany cases, than unconjugated or “naked” antibodies or antibodyfragments, although such unconjugated targeting molecules have been ofuse in specific situations. In cancer, for example, naked antibodieshave come to play a role in the treatment of lymphomas (CAMPATH® andRITUXAN®), colorectal and other cancers (ERBITUX® and AVASTIN®), breastcancer (HERECEPTIN®), as well as a large number now in clinicaldevelopment (e.g., epratuzumab). In most of these cases, clinical usehas involved combining these naked, or unconjugated, antibodies withother therapies, such as chemotherapy or radiation therapy.

A variety of antibodies are also in use for the treatment of autoimmuneand other immune dysregulatory diseases, such as tumor necrosis factor(TNF) and B-cell (RITUXAN®) antibodies in arthritis, and are beinginvestigated in other such diseases, such as the B-cell antibodies,RITUXAN® and epratuzumab, in systemic lupus erythematosus and Sjögren'ssyndrome, as well as juvenile diabetes and multiple sclerosis. Nakedantibodies are also being studied in sepsis and septic shock,Alzheimer's disease, and infectious diseases.

There is a need to develop more potent immunoconjugated antibodiesagainst B cell diseases, such as cancer, autoimmune disease, immunedysfunction disease, type 1 and type 2 diabetes. There is a further needto develop more effective antibody conjugates with intracellularlycleavable linkers. In the case of delivering drug/toxin or radionuclideconjugates, this can be accomplished by direct antibody conjugation orby indirect methods, referred to as pretargeting, where a bispecificantibody is used to target to the lesion, while the therapeutic agent issecondarily targeted by binding to one of the arms of the bispecificantibody that has localized at the site of the diseased cell (Goldenberget al., J Clin Oncol. 2006 Feb. 10; 24(5):823-34.; Goldenberg et al., JNucl Med. 2008 January; 49(1):158-63).

Because signaling pathway redundancies can result in lack of response toa single antibody, diverse strategies to use combination therapy withantibodies that bind to different epitopes or different antigens on thesame target cell have been proposed. Combinations such as anti-CD20 andanti-CD22 (Stein et al., Clin Cancer Res 2004, 10:2868-2878), anti-CD20and anti-HLA-DR (Tobin et al., Leuk Lymphoma 2007, 48:944-956),anti-CD20 and anti-I RAIL-R1 (Maddipatla et al., Clin Cancer Res 2007,13:4556-4564), anti-IGF-1R and anti-EGFR (Goetsche et al., Int J Cancer2005, 113:316-328), anti-IGF-1R and anti-VEGF (Shang et al., Mol CancerTher 2008, 7:2599-2608), or trastuzumab and pertuzumab that targetdifferent regions of human EGFR2 (Nahta et al., Cancer Res 2004,64:2343-2346) have been evaluated preclinically, showing enhanced orsynergistic antitumor activity in vitro and in vivo.

The first clinical evidence of an apparent advantage of combining twoantibodies against different cancer cell antigens involved theadministration of rituximab (chimeric anti-CD20) and epratuzumab(humanized anti-CD22 antibody) in patients with non-Hodgkin lymphoma(NHL). The combination was found to enhance anti-lymphoma efficacywithout a commensurate increase in toxicity, based on 3 independentclinical trials (Leonard et al., J Clin Oncol 2005, 23:5044-5051).Although these results are promising, a need exists in the field formore effective antibody-based combination therapies.

SUMMARY

The present invention concerns compositions and methods of use ofcombination therapy with at least one anti-CD22 antibody or fragmentthereof and one or more therapeutic agents. The therapeutic agent may beselected from the group consisting of an immunomodulator, a cytotoxicagent, a drug, a toxin, an anti-angiogenic agent, a proapoptotic agent,a radionuclide a second antibody or fragment thereof, an siRNA or otherinhibitory oligonucleotide or any other known therapeutic agent.Preferably, the therapeutic agent is conjugated to the anti-CD22antibody or fragment thereof to form an immunoconjugate. However, one ormore additional therapeutic agents, such as a second antibody orfragment thereof, may also be separately administered, either before,simultaneously with or after the immunoconjugate. In most preferredembodiments, the one or more therapeutic agents may comprise acamptothecin, such as SN-38.

Although camptothecin (CPT) and its analogs and derivatives arepreferred chemotherapeutic moieties, other chemotherapeutic agents ofuse may include taxanes (e.g., baccatin III, paclitaxel), epothilones,anthracycline drugs (e.g., doxorubicin, epirubicin,morpholinodoxorubicin, cyanomorpholino-doxorubicin,2-pyrrolinodoxorubicin, see Priebe W (ed.), ACS symposium series 574,American Chemical Society, Washington D.C., 1995; Nagy et al., Proc.Natl. Acad. Sci. USA 93:2464-2469, 1996), benzoquinoid ansamycinsexemplified by geldanamycin (DeBoer et al., Journal of Antibiotics23:442-447, 1970; Neckers et al., Invest. New Drugs 17:361-373, 1999),and the like. Preferably, the antibody is conjugated to at least onechemotherapeutic moiety; preferably 1 to about 5 chemotherapeuticmoieties; most preferably about 6 to 12 chemotherapeutic moieties.

With regard to the CPT group of drugs, issues of insolubility in aqueousbuffers and the lability of the 6-lactone moiety of the E-ring of theirstructures under physiological conditions are relevant. One approach hasbeen to acylate the 20-hydroxyl group with an amino acid, and couple theα-amino group of the amino acid to poly-L-glutamic acid (Singer et al.in The Camptothecins: Unfolding Their Anticancer Potential, Liehr J. G.,Giovanella, B. C. and Verschraegen (eds), NY Acad. Sci., NY 922:136-150,2000). This approach relies on the passive diffusion of a polymericmolecule into tumor sites. This glycine conjugation has also beenreported as a method of making a water-soluble derivative of CPT(Vishnuvajjala et al., U.S. Pat. No. 4,943,579) and in the preparationof a PEG-derivatized CPT (Greenwald, et al. J. Med. Chem. 39: 1938-1940,1996). In the latter case, the approach has been devised in the contextof developing water-soluble and long acting forms of CPT, whereby CPT'sin vivo half-life is enhanced, and the drug is gradually released fromits conjugate while in circulation in vivo. An example of a watersoluble CPT derivative is CPT-11. Extensive clinical data are availableconcerning CPT-11's pharmacology and its in vivo conversion to theactive SN-38 (Iyer and Ratain, Cancer Chemother Pharmacol. 42:S31-43,1998; Mathijssen et al., Clin Cancer Res. 7:2182-2194, 2002; Rivory, AnnNY Acad. Sci. 922:205-215, 2000). The active form SN-38 is about 2 to 3orders of magnitude more potent than CPT-11.

In one embodiment, the invention relates to a process of preparingimmunoconjugates, wherein a drug is derivatized with a first linker,which contains a reactive moiety that is capable of combining with asecond linker that contains an antibody-coupling group; wherein thefirst linker also possesses a defined polyethylene glycol (PEG) moietyfor water-solubility, and optionally an intracellularly-cleavable moietycleavable by intracellular peptidases or by the low pH environment ofendosomal and lysosomal vesicles. Also optionally there is an amino acidspacer between the drug and the first linker. The second linker may alsocontain a reactive group capable of reacting with drug-(first linker)conjugate by the copper (+1) ion-catalyzed acetylene-azide cycloadditionreaction, referred to as ‘click chemistry’. Preferably, the defined PEGmoiety is a low molecular weight PEG with a defined number of monomericsubunits, as discussed below.

Another embodiment relates to a process of preparing conjugates asdiscussed in the paragraph above, wherein the second linker has a singleantibody-coupling group, but multiples of the reactive group capable ofreacting with drug-(first linker) conjugate, thereby amplifying thenumber of drug molecules conjugated to the antibody.

A further embodiment relates to a process of preparing conjugates,wherein the linker is first conjugated to a drug, thereby producing adrug-linker conjugate; wherein said drug-linker conjugate preparationinvolves the selective protection and deprotection of a more reactivegroup in a drug containing multiple functional groups; wherein saiddrug-linker conjugate is optionally not purified; and wherein saiddrug-linker conjugate is subsequently conjugated to a monoclonalantibody or fragment.

In one embodiment, the intracellularly-cleavable moiety is a carbonatecomprising an activated hydroxyl group of the chemotherapeutic moietyand a substituted ethanolamine moiety or a 4-aminobenzyl alcohol, andthe latter is attached, via its amino group, to a cross-linkerterminating in the antibody-binding group; and wherein the substitutedethanolamine moiety is derived from a natural L amino acid, with thecarboxylic acid group of the latter replaced with a hydroxymethylmoiety; and wherein the 4-aminobenzyl alcohol is optionally substitutedwith a C₁-C₁₀ alkyl group at the benzylic position.

In a preferred embodiment, the intracellularly-cleavable moiety is acarbonate comprising an activated hydroxyl group of the chemotherapeuticmoiety and a substituted ethanolamine moiety, and the latter, via itsamino group, is attached to an L-amino acid or a polypeptide comprisingup to four L-amino acid moieties; wherein the N-terminus is attached toa cross-linker terminating in the antibody-binding group; and whereinthe substituted ethanolamine moiety is optionally derived from an Lamino acid, with the carboxylic acid group of the latter replaced with ahydroxymethyl moiety.

In another preferred embodiment, the intracellularly-cleavable moiety isa carbonate comprising an activated hydroxyl group of thechemotherapeutic moiety and a 4-aminobenzyl alcohol or substituted4-aminobenzyl alcohol substituted with a C₁-C₁₀ alkyl group at thebenzylic position, and the latter, via its amino group, is attached toan L-amino acid or a polypeptide comprising up to four L-amino acidmoieties; wherein the N-terminus is attached to a cross-linkerterminating in the antibody-binding group.

In certain embodiments, an amino group of a chemotherapeutic moiety iscoupled to the activated hydroxyl group of a substituted, andamine-protected, ethanolamine moiety or a 4-aminobenzyl alcohol, and thelatter is attached, via its amino group, to an L-amino acid or apolypeptide comprising up to four L-amino acid moieties; wherein theN-terminus is attached to a cross-linker terminating in theantibody-binding group; wherein said substituted ethanolamine moiety isoptionally derived from an L amino acid, with the carboxylic acid groupof the latter replaced with a hydroxymethyl moiety; and wherein the4-aminobenzyl alcohol is optionally substituted with a C₁-C₁₀ alkylgroup at the benzylic position. The bifunctional drug derivative is thenconjugated to an antibody to obtain an immunoconjugate as discussedabove. Upon targeting the disease site with the immunoconjugate, theimmunoconjugate is endocytosed and catabolized to release thedrug-linker moiety; wherein the free amino group of the substitutedethanolamine moiety assists in the liberation of free drug bynucleophilic attack at the carbonyl group of the carbamate moiety.

In certain embodiments, the anti-CD22 antibody or fragment thereof isadministered as part of a trivalent, tetravalent or hexavalent constructmade by the dock-and-lock (DNL) technique (see, e.g., U.S. Pat. Nos.7,521,056; 7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,858,070;7,871,622; 7,901,680; 7,906,118 and 7,906,121, the Examples section ofeach of which is incorporated herein by reference.) The DNL techniquetakes advantage of the specific, high-affinity binding interactionbetween a dimerization and docking domain (DDD) sequence from theregulatory subunit of human cAMP-dependent protein kinase (PKA), such ashuman PKA RIα, RI, RIIα or RII, and an anchor domain (AD) sequence fromany of a variety of AKAP proteins. The DDD and AD peptides may beattached to any protein, peptide or other molecule.

Because the DDD sequences spontaneously dimerize and bind to the ADsequence, the DNL technique allows the formation of complexes betweenany selected molecules that may be attached to DDD or AD sequences.Although the standard DNL complex comprises a trimer with two DDD-linkedmolecules attached to one AD-linked molecule, variations in complexstructure allow the formation of dimers, trimers, tetramers, pentamers,hexamers and other multimers. In some embodiments, the DNL complex maycomprise two or more antibodies, antibody fragments or fusion proteinswhich bind to the same antigenic determinant or to two or more differentantigens. The DNL complex may also comprise one or more other effectors,such as a cytokine, toxin or PEG moiety.

Many examples of anti-CD22 antibodies are also known in the art and anysuch known antibody or fragment thereof may be utilized. In a preferredembodiment, the anti-CD22 antibody is an hLL2 antibody (also known asepratuzumab) that comprises the light chain CDR sequences CDR1(KSSQSVLYSANHKYLA, SEQ ID NO:1), CDR2 (WASTRES, SEQ ID NO:2), and CDR3(HQYLSSWTF, SEQ ID NO:3) and the heavy chain CDR sequences CDR1 (SYWLH,SEQ ID NO:4), CDR2 (YINPRNDYTEYNQNFKD, SEQ ID NO:5), and CDR3 (RDITTFY,SEQ ID NO:6). A humanized LL2 anti-CD22 antibody suitable for use isdisclosed in U.S. Pat. No. 6,187,287, incorporated herein by referencefrom Col. 11, line 40 through Col. 20, line 38 and FIGS. 1, 4 and 5.However, in alternative embodiments, other known and/or commerciallyavailable anti-CD22 antibodies may be utilized, such as 1F5; HIB22(ABBIOTEC®, San Diego, Calif.); FPC1, LT22, MEM-1, RFB4 (ABCAM®,Cambridge, Mass.); bu59, fpc1, mc64-12 (ABD SEROTEC®, Raleigh, N.C.);IS7 (ABNOVA®, Taipei City, Taiwan) and any other anti-CD22 antibodyknown in the art.

The anti-CD22 antibody may be selected such that it competes with orblocks binding to CD22 of an LL2 antibody comprising the light chain CDRsequences CDR1 (KSSQSVLYSANHKYLA, SEQ ID NO:1), CDR2 (WASTRES, SEQ IDNO:2), and CDR3 (HQYLSSW1F, SEQ ID NO:3) and the heavy chain CDRsequences CDR1 (SYWLH, SEQ ID NO:4), CDR2 (YINPRNDYTEYNQNFKD, SEQ IDNO:5), and CDR3 (RDITTFY, SEQ ID NO:6). Alternatively, the anti-CD22antibody may bind to the same epitope of CD22 as an LL2 antibody.

The anti-CD22 antibody may optionally be administered in combinationwith an anti-CD20 antibody or fragment thereof. Many examples ofanti-CD20 antibodies are known in the art and any such known antibody orfragment thereof may be utilized. In a preferred embodiment, theanti-CD20 antibody is an hA20 antibody (also known as veltuzumab) thatcomprises the light chain complementarity-determining region (CDR)sequences CDR1 (RASSSVSYIH; SEQ ID NO:7), CDR2 (ATSNLAS; SEQ ID NO:8),and CDR3 (QQWTSNPPT; SEQ ID NO:9) and the heavy chain variable regionCDR sequences CDR1 (SYNMH; SEQ ID NO:10), CDR2 (AIYPGNGDTSYNQKFKG; SEQID NO:11), and CDR3 (STYYGGDWYFDV; SEQ ID NO:12).

A humanized anti-CD20 antibody suitable for use is disclosed in U.S.Pat. No. 7,435,803, incorporated herein by reference from Col. 36, line4 through Col. 46, line 52 and FIGS. 1, 2, 4, 5 and 7. However, inalternative embodiments, other known and/or commercially availableanti-CD20 antibodies may be utilized, such as rituximab; ofatumumab;ibritumomab; tositumomab; ocrelizumab; GA101; BCX-301; DXL 625; L26,B-Ly1, MEM-97, LT20, 2H7, AT80, B-H20 (ABCAM®, Cambridge, Mass.); HI20a,HI47, 13.6E12 (ABBIOTEC®, San Diego, Calif.); 4f11, 5c11, 7d1 (ABD SERORaleigh, N.C.) and any other anti-CD20 antibody known in the art.

The anti-CD20 antibody may be selected such that it competes with orblocks binding to CD20 of an hA20 antibody comprising the light chaincomplementarity-determining region (CDR) sequences CDR1 (RASSSVSYIH; SEQID NO:7), CDR2 (ATSNLAS; SEQ ID NO:8), and CDR3 (QQWTSNPPT; SEQ ID NO:9)and the heavy chain variable region CDR sequences CDR1 (SYNMH; SEQ IDNO:10), CDR2 (AIYPGNGDTSYNQKFKG; SEQ ID NO:11), and CDR3 (STYYGGDWYFDV;SEQ ID NO:12). Alternatively, the anti-CD20 antibody may bind to thesame epitope of CD20 as a hA20 antibody.

The anti-CD22 antibody may be conjugated to or separately administeredwith one or more therapeutic agents. The therapeutic agent may beselected from the group consisting of aplidin, azaribine, anastrozole,azacytidine, bleomycin, bortezomib, bryostatin-1, busulfan,calicheamycin, camptothecin, 10-hydroxycamptothecin, carmustine,celebrex, chlorambucil, cisplatin, irinotecan (CPT-11), SN-38,carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine,docetaxel, dactinomycin, daunomycin glucuronide, daunorubicin,dexamethasone, diethylstilbestrol, doxorubicin, doxorubicin glucuronide,epirubicin glucuronide, ethinyl estradiol, estramustine, etoposide,etoposide glucuronide, etoposide phosphate, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, fluorouracil,fluoxymesterone, gemcitabine, hydroxyprogesterone caproate, hydroxyurea,idarubicin, ifosfamide, L-asparaginase, leucovorin, lomustine,mechlorethamine, medroprogesterone acetate, megestrol acetate,melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,mithramycin, mitomycin, mitotane, phenyl butyrate, prednisone,procarbazine, paclitaxel, pentostatin, PSI-341, semustine streptozocin,tamoxifen, taxanes, taxol, testosterone propionate, thalidomide,thioguanine, thiotepa, teniposide, topotecan, uracil mustard, velcade,vinblastine, vinorelbine, vincristine, ricin, abrin, ribonuclease,onconase, rapLR1, DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, andPseudomonas endotoxin.

The therapeutic agent may comprise a radionuclide selected from thegroup consisting of ^(103m)Rh, ¹⁰³Ru, ¹⁰⁵Rh, ¹⁰⁵Ru, ¹⁰⁷Hg, ¹⁰⁹Pd, ¹⁰⁹Pt,¹¹¹Ag, ¹¹¹In, ^(113m)In, ¹¹⁹Sb, ¹¹C, ^(121m)Te, ^(122m)Te, ¹²⁵I,^(125m)Te, ¹²⁶I, ¹³¹I, ¹³³I, ¹³N, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵²Dy, ¹⁵³Sm,¹⁵O, ¹⁶¹Ho, ¹⁶¹Tb, ¹⁶⁵Tm, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁶⁹Er, ¹⁶⁹Yb,¹⁷⁷Ln, ¹⁸⁶Re, ¹⁸⁸Re, ^(189m)Os, ^(m189)Re, ¹⁹²Ir, ¹⁹⁴Ir, ¹⁹⁷Pt, ¹⁹⁸Au,¹⁹⁹Au, ²⁰¹Tl, ²⁰³Hg, ²¹¹At, ²¹¹Bi, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²¹⁵Po,²¹⁷At, ²¹⁹Rn, ²²¹Fr, ²²³Ra, ²²⁴Ac, ²²⁵Ac, ²²⁵Fm, ³²P, ³³P, ⁴⁷Sc, ⁵¹Cr,⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶²Cu, ⁶⁷Cu, ⁶⁷Ga, ⁷⁵Br, ⁷⁵Se, ⁷⁶Br, ⁷⁷As, ⁷⁷Br,^(80m)Br, ⁸⁹Sr, ⁹⁰Y, ⁹⁵Ru, ⁹⁷Ru, ⁹⁹Mo and ^(99m)Tc.

The therapeutic agent may be an enzyme selected from the groupconsisting of malate dehydrogenase, staphylococcal nuclease,delta-V-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

The therapeutic agent may be an immunomodulator selected from the groupconsisting of a cytokine, a stem cell growth factor, a lymphotoxin, ahematopoietic factor, a colony stimulating factor (CSF), an interferon(IFN), erythropoietin, thrombopoietin and combinations thereof.Exemplary immunomodulators may include IL-1, IL-2, IL-3, IL-6, IL-10,IL-12, IL-18, IL-21, interferon-α, interferon-β, interferon-γ, G-CSF,GM-CSF, and mixtures thereof.

Alternatively, the therapeutic agent may be an anti-angiogenic agentselected from the group consisting of angiostatin, endostatin,baculostatin, canstatin, maspin, anti-VEGF binding molecules,anti-placental growth factor binding molecules and anti-vascular growthfactor binding molecules.

In certain embodiments, the anti-CD22 antibody or fragment may compriseone or more chelating moieties, such as NOTA, DOTA, DTPA, TETA,Tscg-Cys, or Tsca-Cys. In certain embodiments, the chelating moiety mayform a complex with a therapeutic or diagnostic cation, such as GroupII, Group III, Group IV, Group V, transition, lanthanide or actinidemetal cations, Tc, Re, Bi, Cu, As, Ag, Au, At, or Pb.

In some embodiments, the anti-CD22 antibody or fragment thereof may be ahuman, chimeric, or humanized antibody or fragment thereof. A humanizedantibody or fragment thereof may comprise thecomplementarity-determining regions (CDRs) of a murine antibody and theconstant and framework (FR) region sequences of a human antibody, whichmay be substituted with at least one amino acid from corresponding FRsof a murine antibody. A chimeric antibody or fragment thereof mayinclude the light and heavy chain variable regions of a murine antibody,attached to human antibody constant regions. The antibody or fragmentthereof may include human constant regions of IgG1, IgG2a, IgG3, orIgG4.

Exemplary known antibodies of use include, but are not limited to, hRl(anti-IGF-1R), hPAM4 (anti-mucin), hA20 (anti-CD20), hA19 (anti-CD19),hIMMU31 (anti-AFP), hLL1 (anti-CD74), hLL2 (anti-CD22), hMu-9(anti-CSAp), hL243 (anti-HLA-DR), hMN-14 (anti-CEACAM5), hMN-15(anti-CEACAM6), 29H2 (anti-CEACAM1, ABCAM®), hRS7 (anti-EGP-1) and hMN-3(anti-CEACAM6).

In alternative embodiments, antibodies or fragments of use may bind toone or more target antigens selected from the group consisting ofcarbonic anhydrase IX, alpha-fetoprotein, α-actinin-4, A3, antigenspecific for A33 antibody, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CAl25,CAMEL, CAP-1, CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5,CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25,CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52,CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83,CD95, CD126, CD132, CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A,CXCR4, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM1, CEACAM6,c-met, DAM, EGFR, EGFRvIII, EGP-1, EGP-2, ELF2-M, Ep-CAM, Flt-1, Flt-3,folate receptor, G250 antigen, GAGE, gp100, GROB, HLA-DR, HM1.24, humanchorionic gonadotropin (HCG) and its subunits, HER2/neu, HMGB-1, hypoxiainducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α,IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8,IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growth factor-1(IGF-1), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, LDR/FUT, macrophagemigration inhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2,NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3,MUC4, MUC5, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, antigen specific forPAM-4 antibody, placental growth factor, p53, PLAGL2, prostatic acidphosphatase, PSA, PRAME, PSMA, P1GF, IGF, IGF-1R, IL-6, RS5, RANTES,T101, SAGE, S100, survivin, survivin-2B, TAC, TAG-72, tenascin, FRAILreceptors, TNF-a, Tn antigen, Thomson-Friedenreich antigens, tumornecrosis antigens, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen,complement factors C3, C3a, C3b, C5a, C5, an angiogenesis marker, bcl-2,bcl-6, Kras, cMET, an oncogene marker and an oncogene product (see,e.g., Sensi et al., Clin Cancer Res 2006, 12:5023-32; Parmiani et al., JImmunol 2007, 178:1975-79; Novellino et al. Cancer Immunol Immunother2005, 54:187-207). Reports on tumor associated antigens include Mizukamiet al., (2005, Nature Med. 11:992-97); Hatfield et al., (2005, Curr.Cancer Drug Targets 5:229-48); Vallbohmer et al. (2005, J. Clin. Oncol.23:3536-44); and Ren et al. (2005, Ann. Surg. 242:55-63).

Also disclosed is a method for treating and/or diagnosing a disease ordisorder that includes administering to a patient a therapeutic and/ordiagnostic composition that includes any of the aforementionedantibodies or immunoconjugates or fragments thereof. Typically, thecomposition is administered to the patient intravenously,intramuscularly or subcutaneously at a dose of 20-5000 mg. In preferredembodiments, the disease or disorder is a B-cell lymphoma or leukemia,an immune dysregulation disease, an autoimmune disease, organ-graftrejection or graft-versus-host disease. More preferably, the disease isa B-cell lymphoma or leukemia. Exemplary malignancies that may betreated using the claimed methods and compositions include, but are notlimited to, indolent forms of B-cell lymphomas, aggressive forms ofB-cell lymphomas, acute lymphocytic leukemia, chronic lymphocyticleukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, mantle celllymphoma, diffuse large B-cell lymphoma, follicular lymphoma, marginalzone lymphoma, Burkitt's lymphoma and multiple myeloma

Exemplary autoimmune diseases include acute immune thrombocytopenia,chronic immune thrombocytopenia, dermatomyositis, Sydenham's chorea,myasthenia gravis, systemic lupus erythematosus, lupus nephritis,rheumatic fever, polyglandular syndromes, bullous pemphigoid, pemphigusvulgaris, diabetes mellitus (e.g., juvenile diabetes), Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosum, Takayasu'sarteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitis obliterans, Sjogren's syndrome, primary biliarycirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronicactive hepatitis, polymyositis/dermatomyositis, polychondritis,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis, or fibrosing alveolitis.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Figures are provided to illustrate exemplary, butnon-limiting, preferred embodiments of the invention.

FIG. 1. Preclinical in vivo therapy of athymic nude mice, bearing Capan1 human pancreatic carcinoma, with MAb-CL2A-SN-38 conjugates.

FIG. 2. Preclinical in vivo therapy of athymic nude mice, bearing BxPC3human pancreatic carcinoma, with MAb-CL2A-SN-38 conjugates.

FIG. 3. Preclinical in vivo therapy of athymic nude mice, bearing LS174T human colon carcinoma, with hMN-14-CL2A-SN-38 conjugate.

FIG. 4. Epratuzumab-SN-38 in combation with veltuzumab for treatment offollicular B cell lymphoma (WSU-FSCCL) (Experiment A).

FIG. 5. Epratuzumab-SN-38 in combation with veltuzumab for treatment offollicular B cell lymphoma (WSU-FSCCL) (Experiment B).

FIG. 6. Epratuzumab-SN-38 used alone for treatment of 697 cell line(ALL).

FIG. 7. Dose-response data for epratuzumab-CL2A-SN38 vs. controlMAb-CL2A-SN38 for SC Ramos lymphoma in nude mice.

DETAILED DESCRIPTION Definitions

As used herein, the terms “a”, “an” and “the” may refer to either thesingular or plural, unless the context otherwise makes clear that onlythe singular is meant.

An “antibody” refers to a full-length (i.e., naturally occurring orformed by normal immunoglobulin gene fragment recombinatorial processes)immunoglobulin molecule (e.g., an IgG antibody) or an immunologicallyactive (i.e., antigen-binding) portion of an immunoglobulin molecule,like an antibody fragment.

An “antibody fragment” is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv, single domain antibodies (DABs or VHHs) andthe like, including half-molecules of IgG4 (van der Neut Kolfschoten etal. (Science 2007; 317(14 September):1554-1557). Regardless ofstructure, an antibody fragment binds with the same antigen that isrecognized by the intact antibody. For example, an anti-CD22 antibodyfragment binds with an epitope of CD22. The term “antibody fragment”also includes isolated fragments consisting of the variable regions,such as the “Fv” fragments consisting of the variable regions of theheavy and light chains and recombinant single chain polypeptidemolecules in which light and heavy chain variable regions are connectedby a peptide linker (“scFv proteins”). As used herein, the term“antibody fragment” does not include fragments such as Fc fragments thatdo not contain antigen-binding sites.

A “chimeric antibody” is a recombinant protein that contains thevariable domains including the complementarity determining regions(CDRs) of an antibody derived from one species, preferably a rodentantibody, while the constant domains of the antibody molecule arederived from those of a human antibody. For veterinary applications, theconstant domains of the chimeric antibody may be derived from that ofother species, such as a cat or dog.

A “humanized antibody” is a recombinant protein in which the CDRs froman antibody from one species; e.g., a rodent antibody, are transferredfrom the heavy and light variable chains of the rodent antibody intohuman heavy and light variable domains. Additional FR amino acidsubstitutions from the parent, e.g. murine, antibody may be made. Theconstant domains of the antibody molecule are derived from those of ahuman antibody.

A “human antibody” is, for example, an antibody obtained from transgenicmice that have been genetically engineered to produce human antibodiesin response to antigenic challenge. In this technique, elements of thehuman heavy and light chain locus are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous heavy chain and light chain loci. The transgenic micecan synthesize human antibodies specific for human antigens, and themice can be used to produce human antibody-secreting hybridomas. Methodsfor obtaining human antibodies from transgenic mice are described byGreen et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. (See, e.g., McCafferty et al., Nature 348:552-553 (1990) forthe production of human antibodies and fragments thereof in vitro, fromimmunoglobulin variable domain gene repertoires from unimmunizeddonors). In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see, e.g. Johnsonand Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).Human antibodies may also be generated by in vitro activated B cells.(See, U.S. Pat. Nos. 5,567,610 and 5,229,275).

A “therapeutic agent” is an atom, molecule, or compound that is usefulin the treatment of a disease. Examples of therapeutic agents includebut are not limited to antibodies, antibody fragments, drugs, toxins,enzymes, nucleases, hormones, immunomodulators, antisenseoligonucleotides, chelators, boron compounds, photoactive agents, dyesand radioisotopes.

A “diagnostic agent” is an atom, molecule, or compound that is useful indiagnosing a disease. Useful diagnostic agents include, but are notlimited to, radioisotopes, dyes, contrast agents, fluorescent compoundsor molecules and enhancing agents (e.g., paramagnetic ions). Preferably,the diagnostic agents are selected from the group consisting ofradioisotopes, enhancing agents, and fluorescent compounds.

An “immunoconjugate” is a conjugate of an antibody, antibody fragment,antibody fusion protein, bispecific antibody or multispecific antibodywith an atom, molecule, or a higher-ordered structure (e.g., with acarrier, a therapeutic agent, or a diagnostic agent). A “naked antibody”is an antibody that is not conjugated to any other agent.

As used herein, the term “antibody fusion protein” is a recombinantlyproduced antigen-binding molecule in which an antibody or antibodyfragment is covalently linked to another protein or peptide, such as thesame or different antibody or antibody fragment or a DDD or AD peptide.The fusion protein may comprise a single antibody component, amultivalent or multispecific combination of different antibodycomponents or multiple copies of the same antibody component. The fusionprotein may additionally comprise an antibody or an antibody fragmentand a therapeutic agent. Examples of therapeutic agents suitable forsuch fusion proteins include immunomodulators and toxins. One preferredtoxin comprises a ribonuclease (RNase), preferably a recombinant RNase.

A “multispecific antibody” is an antibody that can bind simultaneouslyto at least two targets that are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and/or an antigen or epitope. A “multivalent antibody” is anantibody that can bind simultaneously to at least two targets that areof the same or different structure. Valency indicates how many bindingarms or sites the antibody has to a single antigen or epitope; i.e.,monovalent, bivalent, trivalent or multivalent. The multivalency of theantibody means that it can take advantage of multiple interactions inbinding to an antigen, thus increasing the avidity of binding to theantigen. Specificity indicates how many antigens or epitopes an antibodyis able to bind; i.e., monospecific, bispecific, trispecific,multispecific. Using these definitions, a natural antibody, e.g., anIgG, is bivalent because it has two binding arms but is monospecificbecause it binds to one epitope. Multispecific, multivalent antibodiesare constructs that have more than one binding site of differentspecificity. For example, a diabody, where one binding site reacts withone antigen and the other with another antigen.

A “bispecific antibody” is an antibody that can bind simultaneously totwo targets which are of different structure.

As used herein, “CPT” is an abbreviation for camptothecin, andrepresents camptothecin itself or an analog or derivative ofcamptothecin. The structures of camptothecin and some of its analogs,with the numbering indicated and the rings labeled with letters A-E, aregiven in formula 1 in Chart 1 below.

CHART 1

Camptothecin Immunoconjugates

Methods are devised in the following ways for the preparation ofimmunoconjugates of chemotherapeutic drugs with antibodies (MAbs). Thedisclosed methods represent a preferred embodiment of the invention. (1)Solubility of the drug may be enhanced by placing a definedpolyethyleneglycol (PEG) moiety (i.e., a PEG containing a defined numberof monomeric units) between the drug and the targeting vector, whereinthe defined PEG is a low molecular weight PEG, preferably containing1-30 monomeric units, more preferably containing 1-12 monomeric units.(2) A first linker connects the drug at one end and terminates with anacetylene or an azide group at the other end. This first linkercomprises a defined PEG moiety with an azide or acetylene group at oneend and a different reactive group, such as carboxylic acid or hydroxylgroup, at the other end. Said bifunctional defined PEG is attached tothe amine group of an amino alcohol, and the hydroxyl group of thelatter is attached to the hydroxyl group on the drug in the form of acarbonate. Alternatively, the non-azide (or acetylene) moiety of saiddefined bifunctional PEG is attached to the N-terminus of an L-aminoacid or a polypeptide, with the C-terminus attached to the amino groupof amino alcohol, and the hydroxy group of the latter is attached to thehydroxyl group of the drug in the form of carbonate or carbamate,respectively. (3) A second linker, comprising an antibody-coupling groupand a reactive group complementary to the azide (or acetylene) group ofthe first linker, namely acetylene (or azide), reacts with thedrug-(first linker) conjugate via acetylene-azide cycloaddition reactionto furnish the final bifunctional drug product that is useful forconjugating to the disease-targeting antibodies. (4) Theantibody-coupling group is designed to be either a thiol or athiol-reactive group. (5) Methods are devised for selective regenerationof the 10-hydroxyl group in the presence of the C-20 carbonate inpreparations of drug-linker precursor involving CPT analogs such asSN-38. (6) Other protecting groups for reactive hydroxyl groups in drugssuch as the phenolic hydroxyl in SN-38, for example, such ast-butyldimethylsilyl or t-butyldiphenylsilyl are also used, and theseare deprotected by tetrabutylammonium fluoride prior to linking of thederivatized drug to a targeting-vector-coupling moiety. (7) The10-hydroxyl group of CPT analogs is alternatively protected as an esteror carbonate, other than ‘BOC’, such that the bifunctional CPT isconjugated to an antibody without prior deprotection of this protectinggroup, and the protecting group is readily deprotected underphysiological pH conditions after the bioconjugate is administered.

In the acetylene-azide coupling, referred to as ‘click chemistry’, theazide part may be on L2 with the acetylene part on L3. Alternatively, L2may contain acetylene, with L3 containing azide. ‘Click chemistry’ is acopper (+1)-catalyzed cycloaddition reaction between an acetylene moietyand an azide moiety, and is a relatively recent technique inbioconjugation (Kolb H C and Sharpless K B, Drug Discov Today 2003; 8:1128-37). Alternative methods of copper-free click chemistry aredescribed below. Click chemistry takes place in aqueous solution atnear-neutral pH conditions, and is thus amenable for drug conjugation.The advantage of click chemistry is that it is chemoselective, andcomplements other well-known conjugation chemistries such as thethiol-maleimide reaction. In the following discussion, where a conjugatecomprises an antibody or antibody fragment, another type of bindingmoiety, such as an aptamer, avimer or targeting peptide, may besubstituted.

An exemplary preferred embodiment is directed to a conjugate of a drugderivative and an antibody of the general formula 2,

MAb-[L2]-[L1]-[AA]_(m)-[A′]-Drug  (2)

where MAb is a disease-targeting antibody, such as an anti-CD22antibody; L2 is a component of the cross-linker comprising anantibody-coupling moiety and one or more of acetylene (or azide) groups;L1 comprises a defined PEG with azide (or acetylene) at one end,complementary to the acetylene (or azide) moiety in L2, and a reactivegroup such as carboxylic acid or hydroxyl group at the other end; AA isan L-amino acid; m is an integer with values of 0, 1, 2, 3, or 4; and A′is an additional spacer, selected from the group of ethanolamine,4-hydroxybenzyl alcohol, 4-aminobenzyl alcohol, or substituted orunsubstituted ethylenediamine. The L amino acids of ‘AA’ are selectedfrom alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine. If the A′ group contains a hydroxyl, it is linkedto the hydroxyl group or amino group of the drug in the form of acarbonate or carbamate, respectively.

In a preferred embodiment of formula 2, A′ is a substituted ethanolaminederived from an L-amino acid, wherein the carboxylic acid group of theamino acid is replaced by a hydroxymethyl moiety. A′ may be derived fromany one of the following L-amino acids: alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine.

In an example of the immunoconjugate of the preferred embodiment offormula 2, m is 0, A′ is L-valinol, and the drug is exemplified bySN-38. The resultant structure is shown in formula 3.

In another example of the immunoconjugate of the preferred embodiment offormula 2, m is 1 and represented by a derivatized L-lysine, A′ isL-valinol, and the drug is exemplified by SN-38. The structure is shownin formula 4.

In this embodiment, an amide bond is first formed between the carboxylicacid of an amino acid such as lysine and the amino group of valinol,using orthogonal protecting groups for the lysine amino groups. Theprotecting group on the N-terminus of lysine is removed, keeping theprotecting group on the side chain of lysine intact, and the N-terminusis coupled to the carboxyl group on the defined PEG with azide (oracetylene) at the other end. The hydroxyl group of valinol is thenattached to the 20-chloroformate derivative of 10-hydroxy-protectedSN-38, and this intermediate is coupled to an L2 component carrying theantibody-binding moiety as well as the complementary acetylene (orazide) group involved in the click cycloaddition chemistry. Finally,removal of protecting groups at both lysine side chain and SN-38 givesthe product of this example, shown in formula 3.

While not wishing to be bound by theory, the small MW SN-38 product,namely valinol-SN-38 carbonate, generated after intracellularproteolysis, has the additional pathway of liberation of intact SN-38through intramolecular cyclization involving the amino group of valinoland the carbonyl of the carbonate.

In another preferred embodiment, A′ of the general formula 2 is A-OH,whereby A-OH is a collapsible moiety such as 4-aminobenzyl alcohol or asubstituted 4-aminobenzyl alcohol substituted with a C₁-C₁₀ alkyl groupat the benzylic position, and the latter, via its amino group, isattached to an L-amino acid or a polypeptide comprising up to fourL-amino acid moieties; wherein the N-terminus is attached to across-linker terminating in the antibody-binding group.

An example of a preferred embodiment is given below, wherein the A-OHembodiment of A′ of general formula (2) is derived from a substituted4-aminobenzyl alcohol, and ‘AA’ is comprised of a single L-amino acidwith m=1 in the general formula (2), and the drug is exemplified withSN-38. The structure is represented below (formula 5, referred to asMAb-CLX-SN-38). The single amino acid AA is selected from any one of thefollowing L-amino acids: alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine. The substituent R on 4-aminobenzylalcohol moiety (A-OH embodiment of A′) is hydrogen or an alkyl groupselected from C₁-C₁₀ alkyl groups.

An embodiment of MAb-CLX-SN-38 of formula 5, wherein the single aminoacid AA is L-lysine and R═H, and the drug is exemplified by SN-38(formula 6; referred to as MAb-CL2A-SN-38).

Other embodiments are possible within the context of10-hydroxy-containing camptothecins, such as SN-38. In the example ofSN-38 as the drug, the more reactive 10-hydroxy group of the drug isderivatized leaving the 20-hydroxyl group unaffected. Within the generalformula 2, A′ is a substituted ethylenediamine. An example of thisembodiment is represented by the formula ‘7’ below, wherein the phenolichydroxyl group of SN-38 is derivatized as a carbamate with a substitutedethylenediamine, with the other amine of the diamine derivatized as acarbamate with a 4-aminobenzyl alcohol, and the latter's amino group isattached to Phe-Lys dipeptide. In this structure (formula 7), R and R′are independently hydrogen or methyl. It is referred to asMAb-CL17-SN-38 or MAb-CL2E-SN-38, when R═R′=methyl.

In a preferred embodiment, AA comprises a polypeptide moiety, preferablya di, tri or tetrapeptide, that is cleavable by intracellular peptidase.Examples are: Ala-Leu, Leu-Ala-Leu, and Ala-Leu-Ala-Leu (SEQ ID NO:13)(Trouet et al., 1982).

In another preferred embodiment, the L1 component of the conjugatecontains a defined polyethyleneglycol (PEG) spacer with 1-30 repeatingmonomeric units. In a further preferred embodiment, PEG is a defined PEGwith 1-12 repeating monomeric units. The introduction of PEG may involveusing heterobifunctionalized PEG derivatives which are availablecommercially. The heterobifunctional PEG may contain an azide oracetylene group. An example of a heterobifunctional defined PEGcontaining 8 repeating monomeric units, with ‘NHS’ being succinimidyl,is given below in formula 8:

In a preferred embodiment, L2 has a plurality of acetylene (or azide)groups, ranging from 2-40, but preferably 2-20, and more preferably 2-5,and a single targeting vector-binding moiety.

A representative SN-38 conjugate of an antibody containing multiple drugmolecules and a single targeting vector-binding moiety is shown below.The ‘L2’ component of this structure is appended to 2 acetylenic groups,resulting in the attachment of two azide-appended SN-38 molecules. Thebonding to MAb is represented as a succinimide.

In preferred embodiments, when the bifunctional drug contains athiol-reactive moiety as the antibody-binding group, the thiols on theantibody are generated on the lysine groups of the antibody using athiolating reagent. Methods for introducing thiol groups onto antibodiesby modifications of MAb's lysine groups are well known in the art (Wongin Chemistry of protein conjugation and cross-linking, CRC Press, Inc.,Boca Raton, Fla. (1991), pp 20-22). Alternatively, mild reduction ofinterchain disulfide bonds on the antibody (Willner et al., BioconjugateChem. 4:521-527 (1993)) using reducing agents such as dithiothreitol(DTT) can generate 7-to-10 thiols on the antibody; which has theadvantage of incorporating multiple drug moieties in the interchainregion of the MAb away from the antigen-binding region.

In a preferred embodiment, the chemotherapeutic moiety is selected fromthe group consisting of doxorubicin (DOX), epirubicin,morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin(cyanomorpholino-DOX), 2-pyrrolino-doxorubicin (2-PDOX), CPT, 10-hydroxycamptothecin, SN-38, topotecan, lurtotecan, 9-aminocamptothecin,9-nitrocamptothecin, taxanes, geldanamycin, ansamycins, and epothilones.In a more preferred embodiment, the chemotherapeutic moiety is SN-38.Preferably, in the conjugates of the preferred embodiments, the antibodylinks to at least one chemotherapeutic moiety; preferably 1 to about 12chemotherapeutic moieties; most preferably about 6 to about 12chemotherapeutic moieties.

Furthermore, in a preferred embodiment, the linker component ‘L2’comprises a thiol group that reacts with a thiol-reactive residueintroduced at one or more lysine side chain amino groups of saidantibody. In such cases, the antibody is pre-derivatized with athiol-reactive group such as a maleimide, vinylsulfone, bromoacetamide,or iodoacetamide by procedures well described in the art.

In the context of these embodiments, a process was surprisinglydiscovered by which CPT drug-linkers can be prepared wherein CPTadditionally has a 10-hydroxyl group. This process involves, but is notlimited to, the protection of the 10-hydroxyl group as at-butyloxycarbonyl (BOC) derivative, followed by the preparation of thepenultimate intermediate of the drug-linker conjugate. Usually, removalof BOC group requires treatment with strong acid such as trifluoroaceticacid (TFA). Under these conditions, the CPT 20-O-linker carbonate,containing protecting groups to be removed, is also susceptible tocleavage, thereby giving rise to unmodified CPT. In fact, the rationalefor using a mildly removable methoxytrityl (MMT) protecting group forthe lysine side chain of the linker molecule, as enunciated in the art,was precisely to avoid this possibility (Walker et al., 2002). It wasdiscovered that selective removal of phenolic BOC protecting group ispossible by carrying out reactions for short durations, optimally 3-to-5minutes. Under these conditions, the predominant product was that inwhich the ‘BOC’ at 10-hydroxyl position was removed, while the carbonateat ‘20’ position was intact.

An alternative approach involves protecting the CPT analog's 10-hydroxyposition with a group other than ‘BOC’, such that the final product isready for conjugation to antibodies without a need for deprotecting the10-OH protecting group. The 10-hydroxy protecting group, which convertsthe 10-OH into a phenolic carbonate or a phenolic ester, is readilydeprotected by physiological pH conditions or by esterases after in vivoadministration of the conjugate. The faster removal of a phenoliccarbonate at the 10 position vs. a tertiary carbonate at the 20 positionof 10-hydroxycamptothecin under physiological condition has beendescribed by He et al. (He et al., Bioorganic & Medicinal Chemistry 12:4003-4008 (2004)). A 10-hydroxy protecting group on SN-38 can be ‘COR’where R can be a substituted alkyl such as “N(CH₃)₂—(CH₂)_(n)—” where nis 1-10 and wherein the terminal amino group is optionally in the formof a quaternary salt for enhanced aqueous solubility, or a simple alkylresidue such as “CH₃—(CH₂)_(n)—” where n is 0-10, or it can be an alkoxymoiety such as “CH₃—(CH₂)_(n)—O—” where n is 0-10, or“N(CH₃)₂—(CH₂)_(n)—O—” where n is 2-10, or“R₁O—(CH₂—CH₂—O)_(n)—CH₂—CH₂—O—” where R₁ is ethyl or methyl and n is aninteger with values of 0-10. These 10-hydroxy derivatives are readilyprepared by treatment with the chloroformate of the chosen reagent, ifthe final derivative is to be a carbonate. Typically, the10-hydroxy-containing camptothecin such as SN-38 is treated with a molarequivalent of the chloroformate in dimethylformamide using triethylamineas the base. Under these conditions, the 20-OH position is unaffected.For forming 10-O-esters, the acid chloride of the chosen reagent isused.

In a preferred process of the preparation of a conjugate of a drugderivative and an antibody of the general formula 2, wherein thedescriptors L2, L1, AA and A-X are as described in earlier sections, thebifunctional drug moiety, [L2]-[L1]-[AA]_(m)-[A-X]-Drug is firstprepared, followed by the conjugation of the bifunctional drug moiety tothe antibody.

In a preferred process of the preparation of a conjugate of a drugderivative and an antibody of the general formula 2, wherein thedescriptors L2, L1, AA and A-OH are as described in earlier sections,the bifunctional drug moiety is prepared by first linking A-OH to theC-terminus of AA via an amide bond, followed by coupling the amine endof AA to a carboxylic acid group of L1. If AA is absent (i.e. m=0), A-OHis directly attached to L1 via an amide bond. The cross-linker,[L1]-[AA]_(m)-[A-OH], is attached to drug's hydroxyl or amino group, andthis is followed by attachment to the L1 moiety, by taking recourse tothe reaction between azide (or acetylene) and acetylene (or azide)groups in L1 and L2 via click chemistry.

In a preferred process of the preparation of a conjugate of a drugderivative and an antibody of the general formula 2, wherein thedescriptors L2, L1, AA and A-OH are as described in earlier sections,the purified conjugate is contained in the pH range of 5.5 to 7.5 in anyof the following Good's biological buffers derived from:2-(N-morpholino)ethanesulfonic acid (MES),N-(2-acetamido)-2-iminodiacetic acid (ADA),1,4-piperazinediethanesulfonic acid (PIPES),N-(2-acetamido)-2-aminoethanesulfonic acid (ACES),N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), andN-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) or HEPES. Themost preferred buffer is 25 mM MES, pH 6.5. In a further preferredprocess of preparation of the specified conjugates, the conjugatesolution is formulated with excipients such as trehalose and polysorbate80 and lyophilized, and the lyophilized preparations are preferablystored at 2-8 deg.

In one embodiment, the antibody is a monoclonal antibody (MAb). In afurther embodiment, the antibody may be a multivalent and/ormultispecific MAb. The antibody may be a murine, chimeric, humanized, orhuman monoclonal antibody, and said antibody may be in intact, fragment(Fab, Fab′, F(ab)₂, F(ab′)₂), or sub-fragment (single-chain constructs)form, or of an IgG1, IgG2a, IgG3, IgG4, IgA isotype, or submoleculestherefrom.

In a preferred embodiment, the intracellularly-cleavable moiety may becleaved after it is internalized into the cell upon binding by theMAb-drug conjugate to a receptor thereof, and particularly cleaved byesterases and peptidases.

Preparation of Antibodies

The complexes described herein may comprise one or more monoclonalantibodies or fragments thereof. Rodent monoclonal antibodies tospecific antigens may be obtained by methods known to those skilled inthe art. (See, e.g., Kohler and Milstein, Nature 256: 495 (1975), andColigan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages2.5.1-2.6.7 (John Wiley & Sons 1991)).

General techniques for cloning murine immunoglobulin variable domainshave been disclosed, for example, by the publication of Orlandi et al.,Proc. Nat'l Acad. Sci. USA 86: 3833 (1989). Techniques for constructingchimeric antibodies are well known to those of skill in the art. As anexample, Leung et al., Hybridoma 13:469 (1994), disclose how theyproduced an LL2 chimera by combining DNA sequences encoding the V_(k)and V_(H) domains of LL2 monoclonal antibody, an anti-CD22 antibody,with respective human and IgG1 constant region domains. This publicationalso provides the nucleotide sequences of the LL2 light and heavy chainvariable regions, V_(k) and V_(H), respectively. Techniques forproducing humanized antibodies are disclosed, for example, by Jones etal., Nature 321: 522 (1986), Riechmann et al., Nature 332: 323 (1988),Verhoeyen et al., Science 239: 1534 (1988), Carter et al., Proc. Nat'lAcad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev. Biotech. 12: 437(1992), and Singer et al., J. Immun. 150: 2844 (1993).

A chimeric antibody is a recombinant protein that contains the variabledomains including the CDRs derived from one species of animal, such as arodent antibody, while the remainder of the antibody molecule; i.e., theconstant domains, is derived from a human antibody. Accordingly, achimeric monoclonal antibody can also be humanized by replacing thesequences of the murine FR in the variable domains of the chimericantibody with one or more different human FR. Specifically, mouse CDRsare transferred from heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. As simply transferring mouse CDRs into human FRs often resultsin a reduction or even loss of antibody affinity, additionalmodification might be required in order to restore the original affinityof the murine antibody. This can be accomplished by the replacement ofone or more some human residues in the FR regions with their murinecounterparts to obtain an antibody that possesses good binding affinityto its epitope. (See, e.g., Tempest et al., Biotechnology 9:266 (1991)and Verhoeyen et al., Science 239: 1534 (1988)).

A fully human antibody can be obtained from a transgenic non-humananimal. (See, e.g., Mendez et al., Nature Genetics, 15: 146-156, 1997;U.S. Pat. No. 5,633,425.) Methods for producing fully human antibodiesusing either combinatorial approaches or transgenic animals transformedwith human immunoglobulin loci are known in the art (e.g., Mancini etal., 2004, New Microbiol. 27:315-28; Conrad and Scheller, 2005, Comb.Chem. High Throughput Screen. 8:117-26; Brekke and Loset, 2003, Curr.Opin. Pharmacol. 3:544-50). Such fully human antibodies are expected toexhibit even fewer side effects than chimeric or humanized antibodiesand to function in vivo as essentially endogenous human antibodies. Incertain embodiments, the claimed methods and procedures may utilizehuman antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40). Human antibodies may be generated from normal humans or fromhumans that exhibit a particular disease state, such as a hematopoieticcancer (Dantas-Barbosa et al., 2005). The advantage to constructinghuman antibodies from a diseased individual is that the circulatingantibody repertoire may be biased towards antibodies againstdisease-associated antigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.) Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.) RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).Library construction was performed according to Andris-Widhopf et al.(2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 1^(st)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.pp. 9.1 to 9.22). The final Fab fragments were digested with restrictionendonucleases and inserted into the bacteriophage genome to make thephage display library. Such libraries may be screened by standard phagedisplay methods. The skilled artisan will realize that this technique isexemplary only and any known method for making and screening humanantibodies or antibody fragments by phage display may be utilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols as discussed above. Methods for obtaining humanantibodies from transgenic mice are described by Green et al., NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal., Int. Immun. 6:579 (1994). A non-limiting example of such a systemis the XENOMOUSE® (e.g., Green et al., 1999, J. Immunol. Methods231:11-23) from Abgenix (Fremont, Calif.). In the XENOMOUSE® and similaranimals, the mouse antibody genes have been inactivated and replaced byfunctional human antibody genes, while the remainder of the mouse immunesystem remains intact.

The XENOMOUSE® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH and Igkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XENOMOUSE®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XENOMOUSE®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XENOMOUSE® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Antibody Cloning and Production

Various techniques, such as production of chimeric or humanizedantibodies, may involve procedures of antibody cloning and construction.The antigen-binding Vic (variable light chain) and V_(H) (variable heavychain) sequences for an antibody of interest may be obtained by avariety of molecular cloning procedures, such as RT-PCR, 5′-RACE, andcDNA library screening. The V genes of an antibody from a cell thatexpresses a murine antibody can be cloned by PCR amplification andsequenced. To confirm their authenticity, the cloned V_(L) and V_(H)genes can be expressed in cell culture as a chimeric Ab as described byOrlandi et al., (Proc. Natl. Acad. Sci., USA, 86: 3833 (1989)). Based onthe V gene sequences, a humanized antibody can then be designed andconstructed as described by Leung et al. (Mol. Immunol., 32: 1413(1995)).

cDNA can be prepared from any known hybridoma line or transfected cellline producing a murine antibody by general molecular cloning techniques(Sambrook et al., Molecular Cloning, A laboratory manual, 2^(nd) Ed(1989)). The Vκ sequence for the antibody may be amplified using theprimers VK1BACK and VK1FOR (Orlandi et al., 1989) or the extended primerset described by Leung et al. (BioTechniques, 15: 286 (1993)). The V_(H)sequences can be amplified using the primer pair VH1BACK/VH1FOR (Orlandiet al., 1989) or the primers annealing to the constant region of murineIgG described by Leung et al. (Hybridoma, 13:469 (1994)). Humanized Vgenes can be constructed by a combination of long oligonucleotidetemplate syntheses and PCR amplification as described by Leung et al.(Mol. Immunol., 32: 1413 (1995)).

PCR products for Vx can be subcloned into a staging vector, such as apBR327-based staging vector, VKpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites. PCR productsfor V_(H) can be subcloned into a similar staging vector, such as thepBluescript-based VHpBS. Expression cassettes containing the Vκ andV_(H) sequences together with the promoter and signal peptide sequencescan be excised from VKpBR and VHpBS and ligated into appropriateexpression vectors, such as pKh and pGlg, respectively (Leung et al.,Hybridoma, 13:469 (1994)). The expression vectors can be co-transfectedinto an appropriate cell and supernatant fluids monitored for productionof a chimeric, humanized or human antibody. Alternatively, the Vκ andV_(H) expression cassettes can be excised and subcloned into a singleexpression vector, such as pdHL2, as described by Gillies et al. (J.Immunol. Methods 125:191 (1989) and also shown in Losman et al., Cancer,80:2660 (1997)).

In an alternative embodiment, expression vectors may be transfected intohost cells that have been pre-adapted for transfection, growth andexpression in serum-free medium. Exemplary cell lines that may be usedinclude the Sp/EEE, Sp/ESF and Sp/ESF-X cell lines (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930 and 7,608,425; the Examples section of each ofwhich is incorporated herein by reference). These exemplary cell linesare based on the Sp2/0 myeloma cell line, transfected with a mutantBcl-EEE gene, exposed to methotrexate to amplify transfected genesequences and pre-adapted to serum-free cell line for proteinexpression.

Antibody Allotypes

Immunogenicity of therapeutic antibodies is associated with increasedrisk of infusion reactions and decreased duration of therapeuticresponse (Baert et al., 2003, N Engl J Med 348:602-08). The extent towhich therapeutic antibodies induce an immune response in the host maybe determined in part by the allotype of the antibody (Stickler et al.,2011, Genes and Immunity 12:213-21). Antibody allotype is related toamino acid sequence variations at specific locations in the constantregion sequences of the antibody. The allotypes of IgG antibodiescontaining a heavy chain γ-type constant region are designated as Gmallotypes (1976, J Immunol 117:1056-59).

For the common IgG1 human antibodies, the most prevalent allotype isG1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21). However, theG1m3 allotype also occurs frequently in Caucasians (Id.). It has beenreported that G1m1 antibodies contain allotypic sequences that tend toinduce an immune response when administered to non-G1m1 (nG1m1)recipients, such as G1m3 patients (Id.). Non-G1m1 allotype antibodiesare not as immunogenic when administered to G1m1 patients (Id.).

The human G1m1 allotype comprises the amino acids D12 (Kabat position356) and L14 (Kabat position 358) in the CH3 sequence of the heavy chainIgG1. The nG1m1 allotype comprises the amino acids E12 and M14 at thesame locations. Both G1m1 and nG1m1 allotypes comprise an E13 residue inbetween the two variable sites and the allotypes are sometimes referredto as DEL and EEM allotypes. A non-limiting example of the heavy chainconstant region sequence for an nG1m1 (G1m3) allotype antibody is shownin Example 1 below for the exemplary antibody veltuzumab (SEQ ID NO:14).

With regard to therapeutic antibodies, veltuzumab (G1m3) and rituximab(G1m17,1) are, respectively, humanized and chimeric IgG1 antibodiesagainst CD20, of use for therapy of a wide variety of hematologicalmalignancies and/or autoimmune diseases. Table 1 compares the allotypesequences of the heavy chain constant region sequences of rituximab vs.veltuzumab. The light chain constant region sequences of the twoantibodies are identical. As shown in Table 1, rituximab (G1m17,1) is aDEL allotype IgG1, with an additional sequence variation at Kabatposition 214 (heavy chain CH1) of lysine in rituximab vs. arginine inveltuzumab. It has been reported that veltuzumab is less immunogenic insubjects than rituximab (see, e.g., Morchhauser et al., 2009, J ClinOncol 27:3346-53; Goldenberg et al., 2009, Blood 113:1062-70; Robak &Robak, 2011, BioDrugs 25:13-25), an effect that has been attributed tothe difference between humanized and chimeric antibodies. However, thedifference in allotypes between the EEM and DEL allotypes likely alsoaccounts for the lower immunogenicity of veltuzumab.

TABLE 1 Allotypes of Rituximab vs. Veltuzumab Heavy chain position andassociated allotypes Complete 214 356/358 431 allotype (allotype)(allotype) (allotype) Rituximab G1m17, 1 K 17 D/L 1 A — Veltuzumab G1m3R 3 E/M — A —

In order to reduce the immunogenicity of therapeutic antibodies inindividuals of nG1m1 genotype, it is desirable to select the allotype ofthe antibody to correspond to the EEM allotype, with a glutamate residueat Kabat position 356, a methionine at Kabat position 358, andpreferably an arginine residue at Kabat position 214. Surprisingly, itwas found that repeated subcutaneous administration of G1m3 antibodiesover a long period of time did not result in a significant immuneresponse.

Known Antibodies

In various embodiments, the claimed methods and compositions may utilizeany of a variety of antibodies known in the art. Antibodies of use maybe commercially obtained from a number of known sources. For example, avariety of antibody secreting hybridoma lines are available from theAmerican Type Culture Collection (ATCC, Manassas, Va.). A large numberof antibodies against various disease targets have been deposited at theATCC and/or have published variable region sequences and are availablefor use in the claimed methods and compositions. See, e.g., U.S. Pat.Nos. 7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509;7,049,060; 7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018;7,037,498; 7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852;6,989,241; 6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813;6,956,107; 6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547;6,921,645; 6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475;6,905,681; 6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594;6,881,405; 6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062;6,861,511; 6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370;6,824,780; 6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450;6,767,711; 6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981;6,730,307; 6,720,155; 6,716,966; 6,709,653; 6,693,176; 6,692,908;6,689,607; 6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734;6,673,344; 6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833;6,610,294; 6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745; 6,572;856; 6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058; 6,528,625;6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915; 6,488,930;6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529; 6,465,173;6,461,823; 6,458,356; 6,455,044; 6,455,040, 6,451,310; 6,444,206,6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726; 6,406,694;6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350; 6,383,759;6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481; 6,355,444;6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571; 6,340,459;6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744; 6,129,914;6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540; 5,814,440;5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595; 5,677,136;5,587,459; 5,443,953, 5,525,338, the Examples section of each of whichis incorporated herein by reference. These are exemplary only and a widevariety of other antibodies and their hybridomas are known in the art.The skilled artisan will realize that antibody sequences orantibody-secreting hybridomas against almost any disease-associatedantigen may be obtained by a simple search of the ATCC, NCBI and/orUSPTO databases for antibodies against a selected disease-associatedtarget of interest. The antigen binding domains of the cloned antibodiesmay be amplified, excised, ligated into an expression vector,transfected into an adapted host cell and used for protein production,using standard techniques well known in the art.

Exemplary known antibodies that may be of use for therapy of cancer orautoimmune disease within the scope of the claimed methods andcompositions include, but are not limited to, LL1 (anti-CD74), LL2 andRH:34 (anti-CD22), RS7 (anti-epithelial glycoprotein-1 (EGP-1)), PAM4and KC4 (both anti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA orCEACAM5, also known as CD66e)), Mu-9 (anti-colon-specific antigen-p),Immu-31 (an anti-alpha-fetoprotein), TAG-72 (e.g., CC49), Tn, J591 orHuJ591 (anti-PSMA (prostate-specific membrane antigen)), AB-PG1-XG1-026(anti-PSMA dimer), D2/B (anti-PSMA), G250 (an anti-carbonic anhydrase IXMAb), hL243 (anti-HLA-DR), R1 (anti-IGF-1R), A20 (anti-CD20), A19(anti-CD19), MN-3 or MN-15 (anti-CEACAM6), alemtuzumab (anti-CD52),bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33),ibritumomab tiuxetan (anti-CD20); panitumumab (anti-EGFR); rituximab(anti-CD20); tositumomab (anti-CD20); GA101 (anti-CD20); and trastuzumab(anti-ErbB2). Such antibodies are known in the art (e.g., U.S. Pat. Nos.5,686,072; 5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104;6,730,300; 6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084;7,238,785; 7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318;7,585,491; 7,612,180; 7,642,239; and U.S. Patent Application Publ. No.20040202666 (now abandoned); 20050271671; and 20060193865; the Examplessection of each incorporated herein by reference.)

Specific known antibodies of use include, but are not limited to, hPAM4(U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No. 7,251,164), hA19 (U.S.Pat. No. 7,109,304), hIMMU31 (U.S. Pat. No. 7,300,655), hLL1 (U.S. Pat.No. 7,312,318,), hLL2 (U.S. Pat. No. 7,074,403), hMu-9 (U.S. Pat. No.7,387,773), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S. Pat. No.6,676,924), hMN-15 (U.S. Pat. No. 7,541,440), hRl (U.S. patentapplication Ser. No. 12/689,336), hRS7 (U.S. Pat. No. 7,238,785), hMN-3(U.S. Pat. No. 7,541,440), 15B8 (anti-CD40, U.S. Pat. No. 7,820,170),AB-PG1-XG1-026 (U.S. patent application Ser. No. 11/983,372, depositedas ATCC PTA-4405 and PTA-4406) and DNB (WO 2009/130575). Other knownantibodies are disclosed, for example, in U.S. Pat. Nos. 5,686,072;5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730.300;6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785;7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318; 7,585,491;7,612,180; 7,642,239; and U.S. Patent Application Publ. No. 20040202666(now abandoned); 20050271671; and 20060193865. The text of each recitedpatent or application is incorporated herein by reference with respectto the Figures and Examples sections.

Anti-TNF-α antibodies are known in the art and may be of use to treatimmune diseases, such as autoimmune disease, immune dysfunction (e.g.,graft-versus-host disease, organ transplant rejection) or diabetes.Known antibodies against TNF-a include the human antibody CDP571 (Ofeiet al., 2011, Diabetes 45:881-85); murine antibodies MTNFα1, M2TNFα1,M3TNFα1, M3TNFAB1, M302B and M303 (Thermo Scientific, Rockford, Ill.);infliximab (Centocor, Malvern, Pa.); certolizumab pegol (UCB, Brussels,Belgium); and adalimumab (Abbott, Abbott Park, Ill.). These and manyother known anti-TNF-a antibodies may be used in the claimed methods andcompositions. Other antibodies of use for therapy of immunedysregulatory or autoimmune disease include, but are not limited to,anti-B-cell antibodies such as veltuzumab, epratuzumab, milatuzumab orhL243; tocilizumab (anti-IL-6 receptor); basiliximab (anti-CD25);daclizumab (anti-CD25); efalizumab (anti-CD11a); GA101 (anti-CD20;Glycart Roche); muromonab-CD3 (anti-CD3 receptor); Benlysta (HumanGenome Sciences); anti-CD40L (UCB, Brussels, Belgium); natalizumab(anti-a4 integrin) and omalizumab (anti-IgE).

Type-1 and Type-2 diabetes may be treated using known antibodies againstB-cell antigens, such as CD22 (epratuzumab), CD74 (milatuzumab), CD19(hA19), CD20 (veltuzumab) or HLA-DR (hL243) (see, e.g., Winer et al.,2011, Nature Med 17:610-18). Anti-CD3 antibodies also have been proposedfor therapy of type 1 diabetes (Cernea et al., 2010, Diabetes Metab Rev26:602-05).

Macrophage migration inhibitory factor (MIF) is an important regulatorof innate and adaptive immunity and apoptosis. It has been reported thatCD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med197:1467-76). The therapeutic effect of antagonistic anti-CD74antibodies on MIF-mediated intracellular pathways may be of use fortreatment of a broad range of disease states, such as cancers of thebladder, prostate, breast, lung, colon and chronic lymphocytic leukemia(e.g., Meyer-Siegler et al., 2004, BMC Cancer 12:34; Shachar & Haran,2011, Leuk Lymphoma 52:1446-54); autoimmune diseases such as rheumatoidarthritis and systemic lupus erythematosus (Morand & Leech, 2005, FrontBiosci 10:12-22; Shachar & Haran, 2011, Leuk Lymphoma 52:1446-54);kidney diseases such as renal allograft rejection (Lan, 2008, NephronExp Nephrol. 109:e79-83); and numerous inflammatory diseases(Meyer-Siegler et al., 2009, Mediators Inflamm epub Mar. 22, 2009;Takahashi et al., 2009, Respir Res 10:33; Milatuzumab (hLL1) is anexemplary anti-CD74 antibody of therapeutic use for treatment ofMIF-mediated diseases.

Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. The antibody fragments are antigen binding portions ofan antibody, such as F(ab)₂, Fab′, Fab, Fv, scFv and the like. Otherantibody fragments include, but are not limited to, F(ab′)₂ fragmentswhich can be produced by pepsin digestion of the antibody molecule andFab′ fragments which can be generated by reducing disulfide bridges ofthe F(ab′)₂ fragments. Alternatively, Fab′ expression libraries can beconstructed (Huse et al., 1989, Science, 246:1274-1281) to allow rapidand easy identification of monoclonal Fab′ fragments with the desiredspecificity.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). Methodsfor making scFv molecules and designing suitable peptide linkers aredisclosed in U.S. Pat. No. 4,704,692, U.S. Pat. No. 4,946,778, R. Raagand M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80 (1995) and R. E.Bird and B. W. Walker, “Single Chain Antibody Variable Regions,”TIBTECH, Vol 9: 132-137 (1991).

An antibody fragment can be prepared by known methods, for example, asdisclosed by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647 andreferences contained therein. Also, see Nisonoff et al., Arch Biochem.Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119 (1959), Edelman etal., in METHODS IN ENZYMOLOGY VOL.1, page 422 (Academic Press 1967), andColigan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.

A single complementarity-determining region (CDR) is a segment of thevariable region of an antibody that is complementary in structure to theepitope to which the antibody binds and is more variable than the restof the variable region. Accordingly, a CDR is sometimes referred to ashypervariable region. A variable region comprises three CDRs. CDRpeptides can be obtained by constructing genes encoding the CDR of anantibody of interest. Such genes are prepared, for example, by using thepolymerase chain reaction to synthesize the variable region from RNA ofantibody-producing cells. (See, e.g., Larrick et al., Methods: ACompanion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck,“Genetic Manipulation of Monoclonal Antibodies,” in MONOCLONALANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter etal. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward etal., “Genetic Manipulation and Expression of Antibodies,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al., (eds.), pages137-185 (Wiley-Liss, Inc. 1995).

Another form of an antibody fragment is a single-domain antibody (dAb),sometimes referred to as a single chain antibody. Techniques forproducing single-domain antibodies are well known in the art (see, e.g.,Cossins et al., Protein Expression and Purification, 2007, 51:253-59;Shuntao et al., Molec Immunol 2006, 43:1912-19; Tanha et al., J. Biol.Chem. 2001, 276:24774-780). Single domain antibodies may be obtained,for example, from camels, alpacas or llamas by standard immunizationtechniques. (See, e.g., Muyldermans et al., TIBS 26:230-235, 2001; Yauet al., J Immunol Methods 281:161-75, 2003; Maass et al., J ImmunolMethods 324:13-25, 2007). They can have potent antigen-binding capacityand can interact with novel epitopes that are inaccessible toconventional V_(H)-V_(L) pairs. (Muyldermans et al., 2001). Alpaca serumIgG contains about 50% camelid heavy chain only IgG antibodies (HCAbs)(Maass et al., 2007). Alpacas may be immunized with known antigens, suchas TNF-α, and single domain antibodies can be isolated that bind to andneutralize the target antigen (Maass et al., 2007). PCR primers thatamplify virtually all alpaca antibody coding sequences have beenidentified and may be used to construct single domain phage displaylibraries, which can be used for antibody fragment isolation by standardbiopanning techniques well known in the art (Maass et al., 2007).

In certain embodiments, the sequences of antibodies or antibodyfragments, such as the Fc portions of antibodies, may be varied tooptimize their physiological characteristics, such as the half-life inserum. Methods of substituting amino acid sequences in proteins arewidely known in the art, such as by site-directed mutagenesis (e.g.Sambrook et al., Molecular Cloning, A laboratory manual, 2^(nd) Ed,1989). In preferred embodiments, the variation may involve the additionor removal of one or more glycosylation sites in the Fc sequence (e.g.,U.S. Pat. No. 6,254,868, the Examples section of which is incorporatedherein by reference). In other preferred embodiments, specific aminoacid substitutions in the Fc sequence may be made (e.g., Hornick et al.,2000, J Nucl Med 41:355-62; Hinton et al., 2006, J Immunol 176:346-56;Petkova et al. 2006, Int Immunol 18:1759-69; U.S. Pat. No. 7,217,797).

Multispecific and Multivalent Antibodies

Various embodiments may concern use of multispecific and/or multivalentantibodies. For example, an anti-CD22 antibody or fragment thereof andan anti-CD20 antibody or fragment thereof may be joined together bymeans such as the dock-and-lock technique described above. Othercombinations of antibodies or fragments thereof may be utilized. Forexample, the anti-CD22 antibody could be combined with another antibodyagainst a different epitope of the same antigen, or alternatively withan antibody against another antigen, such as CD4, CD5, CD8, CD14, CD15,CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46,CD52, CD54, CD74, CD80, CD126, CD138, B7, HM1.24, HLA-DR, anangiogenesis factor, tenascin, VEGF, P1GF, ED-B fibronectin, anoncogene, an oncogene product, NCA 66a-d, necrosis antigens, Ii (HLA-DRinvariant chain), IL-2, T101, TAC, IL-6, MUC-1, TRAIL-R1 (DR4) orTRAIL-R2 (DR5).

Methods for producing bispecific antibodies include engineeredrecombinant antibodies which have additional cysteine residues so thatthey crosslink more strongly than the more common immunoglobulinisotypes. (See, e.g., FitzGerald et al, Protein Eng 10:1221-1225, 1997).Another approach is to engineer recombinant fusion proteins linking twoor more different single-chain antibody or antibody fragment segmentswith the needed dual specificities. (See, e.g., Coloma et al., NatureBiotech. 15:159-163, 1997). A variety of bispecific antibodies can beproduced using molecular engineering. In one form, the bispecificantibody may consist of, for example, a scFv with a single binding sitefor one antigen and a Fab fragment with a single binding site for asecond antigen. In another form, the bispecific antibody may consist of,for example, an IgG with two binding sites for one antigen and two scFvwith two binding sites for a second antigen.

Dock-and-Lock (DNL)

In preferred embodiments, multivalent monospecific or bispecificantibodies may be produced using the dock-and-lock (DNL) technology(see, e.g., U.S. Pat. Nos. 7,521,056; 7,550,143; 7,534,866; 7,527,787and 7,666,400; the Examples section of each of which is incorporatedherein by reference). The DNL method exploits specific protein/proteininteractions that occur between the regulatory (R) subunits ofcAMP-dependent protein kinase (PKA) and the anchoring domain (AD) ofA-kinase anchoring proteins (AKAPs) (Baillie et al., FEBS Letters. 2005;579: 3264. Wong and Scott, Nat. Rev. Mol. Cell. Biol. 2004; 5: 959).PKA, which plays a central role in one of the best studied signaltransduction pathways triggered by the binding of the second messengercAMP to the R subunits, was first isolated from rabbit skeletal musclein 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure ofthe holoenzyme consists of two catalytic subunits held in an inactiveform by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443). Isozymesof PKA are found with two types of R subunits (RI and RH), and each typehas α and β isoforms (Scott, Pharmacol. Ther. 1991; 50:123). Thus, thereare four types of PKA regulatory subunits—RIα, RIβ, RIIα and RIIβ. The Rsubunits have been isolated only as stable dimers and the dimerizationdomain has been shown to consist of the first 44 amino-terminal residues(Newlon et al., Nat. Struct. Biol. 1999; 6:222). Binding of cAMP to theR subunits leads to the release of active catalytic subunits for a broadspectrum of serine/threonine kinase activities, which are orientedtoward selected substrates through the compartmentalization of PKA viaits docking with AKAPs (Scott et al., J. Biol. Chem. 1990; 265; 21561).

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci. USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell. Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). AKAPs will only bind to dimeric R subunits. For humanRIIα, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human PKAregulatory subunit and the AD of AKAP as an excellent pair of linkermodules for docking any two entities, referred to hereafter as A and B,into a noncovalent complex, which could be further locked into a stablytethered structure through the introduction of cysteine residues intoboth the DDD and AD at strategic positions to facilitate the formationof disulfide bonds. The general methodology of the “dock-and-lock”approach is as follows. Entity A is constructed by linking a DDDsequence to a precursor of A, resulting in a first component hereafterreferred to as a. Because the DDD sequence would effect the spontaneousformation of a dimer, A would thus be composed of a₂. Entity B isconstructed by linking an AD sequence to a precursor of B, resulting ina second component hereafter referred to as b. The dimeric motif of DDDcontained in a₂ will create a docking site for binding to the ADsequence contained in b, thus facilitating a ready association of a₂ andb to form a binary, trimeric complex composed of a₁b. This binding eventis made irreversible with a subsequent reaction to covalently secure thetwo entities via disulfide bridges, which occurs very efficiently basedon the principle of effective local concentration because the initialbinding interactions should bring the reactive thiol groups placed ontoboth the DDD and AD into proximity (Chmura et al., Proc. Natl. Acad.Sci. USA. 2001; 98:8480) to ligate site-specifically. Using variouscombinations of linkers, adaptor modules and precursors, a wide varietyof DNL constructs of different stoichiometry may be produced and used,including but not limited to dimeric, trimeric, tetrameric, pentamericand hexameric DNL constructs (see, e.g., U.S. Pat. Nos. 7,550,143;7,521,056; 7,534,866; 7,527,787 and 7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNLconstruct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the parts) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

The skilled artisan will realize that the DNL technique may be utilizedto produce complexes comprising multiple copies of the same anti-CD22 oranti-CD20 antibodies, or to attach one or more anti-CD22 antibodies toone or more anti-CD20 antibodies or an antibody against a differenttarget antigen expressed by B-cells. Alternatively, the DNL techniquemay be used to attach antibodies to different effector moieties, such astoxins, cytokines, carrier proteins for siRNA and other known effectors.

Structure-Function Relationships in AD and DDD Moieties

For different types of DNL constructs, different AD or DDD sequences maybe utilized. Exemplary DDD and AD sequences are provided below.

DDD1 (SEQ ID NO: 15) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 16) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1(SEQ ID NO: 17) QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 18)CGQIEYLAKQIVDNAIQQAGC

The skilled artisan will realize that DDD1 and DDD2 are based on the DDDsequence of the human RIIα isoform of protein kinase A. However, inalternative embodiments, the DDD and AD moieties may be based on the DDDsequence of the human Ma form of protein kinase A and a correspondingAKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3 (SEQ ID NO: 19) SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEA KDDD3C (SEQ ID NO: 20) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK AD3 (SEQ ID NO: 21) CGFEELAWKIAKMIWSDVFQQGC

In other alternative embodiments, other sequence variants of AD and/orDDD moieties may be utilized in construction of the DNL complexes. Forexample, there are only four variants of human PKA DDD sequences,corresponding to the DDD moieties of PKA RIα, RIIα, RIβ and RIIβ. TheRIIα DDD sequence is the basis of DDD1 and DDD2 disclosed above. Thefour human PKA DDD sequences are shown below. The DDD sequencerepresents residues 1-44 of RIIα, 1-44 of RIIβ, 12-61 of RIα and 13-66of RIβ. (Note that the sequence of DDD1 is modified slightly from thehuman PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 22)SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEE AK PKA RIβ(SEQ ID NO: 23) SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEEN RQILAPKA RIIα (SEQ ID NO: 24) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQPKA RIIβ (SEQ ID NO: 25) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38;Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker etal., 2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al.,2006, Mol Cell 24:397-408.)

For example, Kinderman et al. (2006, Mol Cell 24:397-408) examined thecrystal structure of the AD-DDD binding interaction and concluded thatthe human DDD sequence contained a number of conserved amino acidresidues that were important in either dimer formation or AKAP binding,underlined in SEQ ID NO:15 below. (See FIG. 1 of Kinderman et al., 2006,incorporated herein by reference.) The skilled artisan will realize thatin designing sequence variants of the DDD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical fordimerization and AKAP binding.

(SEQ ID NO: 15) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

As discussed in more detail below, conservative amino acid substitutionshave been characterized for each of the twenty common L-amino acids.Thus, based on the data of Kinderman (2006) and conservative amino acidsubstitutions, potential alternative DDD sequences based on SEQ ID NO:15are shown in Table 2. In devising Table 2, only highly conservativeamino acid substitutions were considered. For example, charged residueswere only substituted for residues of the same charge, residues withsmall side chains were substituted with residues of similar size,hydroxyl side chains were only substituted with other hydroxyls, etc.Because of the unique effect of proline on amino acid secondarystructure, no other residues were substituted for proline. Even withsuch conservative substitutions, there are over twenty million possiblealternative sequences for the 44 residue peptide(2×3×2×2×2×2×2×2×2×2×2×2×2×2×2×4×2×2×2×2×2×4×2×4). A limited number ofsuch potential alternative DDD moiety sequences are shown in SEQ IDNO:26 to SEQ ID NO:45 below. The skilled artisan will realize that analmost unlimited number of alternative species within the genus of DDDmoieties can be constructed by standard techniques, for example using acommercial peptide synthesizer or well known site-directed mutagenesistechniques. The effect of the amino acid substitutions on AD moietybinding may also be readily determined by standard binding assays, forexample as disclosed in Alto et al. (2003, Proc Natl Acad Sci USA100:4445-50).

TABLE 2 Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO: 15). Consensus sequence disclosed as SEQ ID NO: 131.

THIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 26)SKIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 27)SRIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 28)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 29)SHIQIPPALTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 30)SHIQIPPGLSELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 31)SHIQIPPGLTDLLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 32)SHIQIPPGLTELLNGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 33)SHIQIPPGLTELLQAYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 34)SHIQIPPGLTELLQGYSVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 35)SHIQIPPGLTELLQGYTVDVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 36)SHIQIPPGLTELLQGYTVEVLKQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 37)SHIQIPPGLTELLQGYTVEVLRNQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 38)SHIQIPPGLTELLQGYTVEVLRQNPPDLVEFAVEYFTRLREARA (SEQ ID NO: 39)SHIQIPPGLTELLQGYTVEVLRQQPPELVEFAVEYFTRLREARA (SEQ ID NO: 40)SHIQIPPGLTELLQGYTVEVLRQQPPDLVDFAVEYP1RLREARA (SEQ ID NO: 41)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFLVEYFTRLREARA (SEQ ID NO: 42)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFIVEYFTRLREARA (SEQ ID NO: 43)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFVVEYFTRLREARA (SEQ ID NO: 44)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVDYFIRLREARA (SEQ ID NO: 45)

Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50) performed abioinformatic analysis of the AD sequence of various AKAP proteins todesign an RII selective AD sequence called AKAP-IS (SEQ ID NO:17), witha binding constant for DDD of 0.4 nM. The AKAP-IS sequence was designedas a peptide antagonist of AKAP binding to PKA. Residues in the AKAP-ISsequence where substitutions tended to decrease binding to DDD areunderlined in SEQ ID NO:17 below. The skilled artisan will realize thatin designing sequence variants of the AD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical forDDD binding. Table 3 shows potential conservative amino acidsubstitutions in the sequence of AKAP-IS (AD1, SEQ ID NO:17), similar tothat shown for DDD1 (SEQ ID NO:15) in Table 2 above.

Even with such conservative substitutions, there are over thirty-fivethousand possible alternative sequences for the 17 residue AD1 (SEQ IDNO:17) peptide sequence (2×3×2×4×3×2×2×2×2×2×2×4). A limited number ofsuch potential alternative AD moiety sequences are shown in SEQ ID NO:46to SEQ ID NO:63 below. Again, a very large number of species within thegenus of possible AD moiety sequences could be made, tested and used bythe skilled artisan, based on the data of Alto et al. (2003). It isnoted that FIG. 2 of Alto (2003) shows an even large number of potentialamino acid substitutions that may be made, while retaining bindingactivity to DDD moieties, based on actual binding experiments.

AKAP-IS (SEQ ID NO: 17) QIEYLAKQIVDNAIQQA

TABLE 3 Conservative Amino Acid Substitutions in AD1 (SEQ ID NO: 17).Consensus sequence disclosed as SEQ ID NO: 132.

NIEYLAKQIVDNAIQQA (SEQ ID NO: 46) QLEYLAKQIVDNAIQQA (SEQ ID NO: 47)QVEYLAKQIVDNAIQQA (SEQ ID NO: 48) QIDYLAKQIVDNAIQQA (SEQ ID NO: 49)QIEFLAKQIVDNAIQQA (SEQ ID NO: 50) QIETLAKQIVDNAIQQA (SEQ ID NO: 51)QIESLAKQIVDNAIQQA (SEQ ID NO: 52) QIEYIAKQIVDNAIQQA (SEQ ID NO: 53)QIEYVAKQIVDNAIQQA (SEQ ID NO: 54) QIEYLARQIVDNAIQQA (SEQ ID NO: 55)QIEYLAKNIVDNAIQQA (SEQ ID NO: 56) QIEYLAKQIVENAIQQA (SEQ ID NO: 57)QIEYLAKQIVDQAIQQA (SEQ ID NO: 58) QIEYLAKQIVDNAINQA (SEQ ID NO: 59)QIEYLAKQIVDNAIQNA (SEQ ID NO: 60) QIEYLAKQIVDNAIQQL (SEQ ID NO: 61)QIEYLAKQIVDNAIQQI (SEQ ID NO: 62) QIEYLAKQIVDNAIQQV (SEQ ID NO: 63)

Gold et al. (2006, Mol Cell 24:383-95) utilized crystallography andpeptide screening to develop a SuperAKAP-IS sequence (SEQ ID NO:64),exhibiting a five order of magnitude higher selectivity for the RIIisoform of PKA compared with the RI isoform. Underlined residuesindicate the positions of amino acid substitutions, relative to theAKAP-IS sequence, which increased binding to the DDD moiety of RIIα. Inthis sequence, the N-terminal Q residue is numbered as residue number 4and the C-terminal A residue is residue number 20. Residues wheresubstitutions could be made to affect the affinity for RIIα wereresidues 8, 11, 15, 16, 18, 19 and 20 (Gold et al., 2006). It iscontemplated that in certain alternative embodiments, the SuperAKAP-ISsequence may be substituted for the AKAP-IS AD moiety sequence toprepare DNL constructs. Other alternative sequences that might besubstituted for the AKAP-IS AD sequence are shown in SEQ ID NO:65-67.Substitutions relative to the AKAP-IS sequence are underlined. It isanticipated that, as with the AD2 sequence shown in SEQ ID NO:18, the ADmoiety may also include the additional N-terminal residues cysteine andglycine and C-terminal residues glycine and cysteine.

SuperAKAP-IS (SEQ ID NO: 64) QIEYVAKQIVDYAIHQAAlternative AKAP sequences (SEQ ID NO: 65) QIEYKAKQIVDHAIHQA(SEQ ID NO: 66) QIEYHAKQIVDHAIHQA (SEQ ID NO: 67) QIEYVAKQIVDHAIHQA

FIG. 2 of Gold et al. disclosed additional DDD-binding sequences from avariety of AKAP proteins, shown below.

RII-Specific AKAPs

AKAP-KL (SEQ ID NO: 68) PLEYQAGLLVQNAIQQAI AKAP79 (SEQ ID NO: 69)LLIETASSLVKNAIQLSI AKAP-Lbc (SEQ ID NO: 70) LIEEAASRIVDAVIEQVK

RI-Specific AKAPs

AKAPce (SEQ ID NO: 71) ALYQFADRFSELVISEAL RIAD (SEQ ID NO: 72)LEQVANQLADQIIKEAT PV38 (SEQ ID NO: 73) FEELAWKIAKMIWSDVF

Dual-Specificity AKAPs

AKAP7 (SEQ ID NO: 74) ELVRLSKRLVENAVLKAV MAP2D (SEQ ID NO: 75)TAEEVSARIVQVVTAEAV DAKAP1 (SEQ ID NO: 76) QIKQAAFQLISQVILEAT DAKAP2(SEQ ID NO: 77) LAWKIAKMIVSDVMQQ

Stokka et al. (2006, Biochem J 400:493-99) also developed peptidecompetitors of AKAP binding to PKA, shown in SEQ ID NO:78-80. Thepeptide antagonists were designated as Ht31 (SEQ ID NO:78), RIAD (SEQ IDNO:79) and PV-38 (SEQ ID NO:80). The Ht-31 peptide exhibited a greateraffinity for the RH isoform of PKA, while the RIAD and PV-38 showedhigher affinity for R1.

Ht31 (SEQ ID NO: 78) DLIEEAASRIVDAVIEQVKAAGAY RIAD (SEQ ID NO: 79)LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 80) FEELAWKIAKMIWSDVFQQC

Hundsrucker et al. (2006, Biochem J 396:297-306) developed still otherpeptide competitors for AKAP binding to PKA, with a binding constant aslow as 0.4 nM to the DDD of the RII form of PKA. The sequences ofvarious AKAP antagonistic peptides are provided in Table 1 ofHundsrucker et al., reproduced in Table 4 below. AKAPIS represents asynthetic RII subunit-binding peptide. All other peptides are derivedfrom the RII-binding domains of the indicated AKAPs.

TABLE 4 AKAP Peptide sequences Peptide Sequence AKAPIS QIEYLAKQIVDNAIQQA(SEQ ID NO: 17) AKAPIS-P QIEYLAKQIPDNAIQQA (SEQ ID NO: 81) Ht31KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 82) Ht31-PKGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 83) AKAP7δ-PEDAELVRLSKRLVENAVLKAVQQY wt-pep (SEQ ID NO: 84) AKAP7δ-PEDAELVRTSKRLVENAVLKAVQQY L304T-pep (SEQ ID NO: 85) AKAP7δ-PEDAELVRLSKRDVENAVLKAVQQY L308D-pep (SEQ ID NO: 86) AKAP7δ-PEDAELVRLSKRLPENAVLKAVQQY P-pep (SEQ ID NO: 87) AKAP7δ-PEDAELVRLSKRLPENAPLKAVQQY PP-pep (SEQ ID NO: 88) AKAP7δ-PEDAELVRLSKRLVENAVEKAVQQY L314E-pep (SEQ ID NO: 89) AKAP1-pepEEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 90) AKAP2-pepLVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 91) AKAP5-pepQYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 92) AKAP9-pepLEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 93) AKAP10-pepNTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 94) AKAP11-pepVNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 95) AKAP12-pepNGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 96) AKAP14-pepTQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 97) Rab32-pepETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 98)

Residues that were highly conserved among the AD domains of differentAKAP proteins are indicated below by underlining with reference to theAKAP IS sequence (SEQ ID NO:17). The residues are the same as observedby Alto et al. (2003), with the addition of the C-terminal alanineresidue. (See FIG. 4 of Hundsrucker et al. (2006), incorporated hereinby reference.) The sequences of peptide antagonists with particularlyhigh affinities for the RII DDD sequence were those of AKAP-IS,AKAP7δ-wt-pep, AKAP7δ-L304T-pep and AKAP7δ-L308D-pep.

AKAP-IS (SEQ ID NO: 17) QIEYLAKQIVDNAIQQA

Carr et al. (2001, J Biol Chem 276:17332-38) examined the degree ofsequence homology between different AKAP-binding DDD sequences fromhuman and non-human proteins and identified residues in the DDDsequences that appeared to be the most highly conserved among differentDDD moieties. These are indicated below by underlining with reference tothe human PKA RIIα DDD sequence of SEQ ID NO:15. Residues that wereparticularly conserved are further indicated by italics. The residuesoverlap with, but are not identical to those suggested by Kinderman etal. (2006) to be important for binding to AKAP proteins. The skilledartisan will realize that in designing sequence variants of DDD, itwould be most preferred to avoid changing the most conserved residues(italicized), and it would be preferred to also avoid changing theconserved residues (underlined), while conservative amino acidsubstitutions may be considered for residues that are neither underlinednor italicized.

(SEQ ID NO: 15) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR LREA R A

A modified set of conservative amino acid substitutions for the DDD1(SEQ ID NO:15) sequence, based on the data of Can et al. (2001) is shownin Table 5. Even with this reduced set of substituted sequences, thereare over 65,000 possible alternative DDD moiety sequences that may beproduced, tested and used by the skilled artisan without undueexperimentation. The skilled artisan could readily derive suchalternative DDD amino acid sequences as disclosed above for Table 2 andTable 3.

TABLE 5 Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO: 15).Consensus sequence disclosed as SEQ ID NO: 133.

The skilled artisan will realize that these and other amino acidsubstitutions in the DDD or AD amino acid sequences may be utilized toproduce alternative species within the genus of AD or DDD moieties,using techniques that are standard in the field and only routineexperimentation.

Amino Acid Substitutions

In various embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. For example, the DDD and/or ADsequences used to make DNL constructs may be modified as discussedabove.

The skilled artisan will be aware that, in general, amino acidsubstitutions typically involve the replacement of an amino acid withanother amino acid of relatively similar properties (i.e., conservativeamino acid substitutions). The properties of the various amino acids andeffect of amino acid substitution on protein structure and function havebeen the subject of extensive study and knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+-0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (O) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,H is, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Immunoconjugates

In certain embodiments, an antibody or antibody fragment may be directlyattached to one or more therapeutic agents to form an immunoconjugate.Therapeutic agents may be attached, for example to reduced SH groupsand/or to carbohydrate side chains. A therapeutic agent can be attachedat the hinge region of a reduced antibody component via disulfide bondformation. Alternatively, such agents can be attached using aheterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). Alternatively, the therapeutic agentcan be conjugated via a carbohydrate moiety in the Fc region of theantibody.

Methods for conjugating functional groups to antibodies via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, the Examples section of which is incorporated herein byreference. The general method involves reacting an antibody having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function. This reaction results in an initial Schiff base(imine) linkage, which can be stabilized by reduction to a secondaryamine to form the final conjugate.

The Fc region may be absent if the antibody component of theimmunoconjugate is an antibody fragment. However, it is possible tointroduce a carbohydrate moiety into the light chain variable region ofa full length antibody or antibody fragment. See, for example, Leung etal., J. Immunol. 154: 5919 (1995); U.S. Pat. Nos. 5,443,953 and6,254,868, the Examples section of which is incorporated herein byreference. The engineered carbohydrate moiety is used to attach thetherapeutic or diagnostic agent.

An alternative method for attaching therapeutic agents to an antibody orfragment involves use of click chemistry reactions. The click chemistryapproach was originally conceived as a method to rapidly generatecomplex substances by joining small subunits together in a modularfashion. (See, e.g., Kolb et al., 2004, Angew Chem Int Ed 40:3004-31;Evans, 2007, Aust J Chem 60:384-95.) Various forms of click chemistryreaction are known in the art, such as the Huisgen 1,3-dipolarcycloaddition copper catalyzed reaction (Tornoe et al., 2002, J OrganicChem 67:3057-64), which is often referred to as the “click reaction.”Other alternatives include cycloaddition reactions such as theDiels-Alder, nucleophilic substitution reactions (especially to smallstrained rings like epoxy and aziridine compounds), carbonyl chemistryformation of urea compounds and reactions involving carbon-carbon doublebonds, such as alkynes in thiol-yne reactions.

The azide alkyne Huisgen cycloaddition reaction uses a copper catalystin the presence of a reducing agent to catalyze the reaction of aterminal alkyne group attached to a first molecule. In the presence of asecond molecule comprising an azide moiety, the azide reacts with theactivated alkyne to form a 1,4-disubstituted 1,2,3-triazole. The coppercatalyzed reaction occurs at room temperature and is sufficientlyspecific that purification of the reaction product is often notrequired. (Rostovstev et al., 2002, Angew Chem Int Ed 41:2596; Tornoe etal., 2002, J Org Chem 67:3057.) The azide and alkyne functional groupsare largely inert towards biomolecules in aqueous medium, allowing thereaction to occur in complex solutions. The triazole formed ischemically stable and is not subject to enzymatic cleavage, making theclick chemistry product highly stable in biological systems. Althoughthe copper catalyst is toxic to living cells, the copper-based clickchemistry reaction may be used in vitro for immunoconjugate formation.

A copper-free click reaction has been proposed for covalent modificationof biomolecules. (See, e.g., Agard et al., 2004, J Am Chem Soc126:15046-47.) The copper-free reaction uses ring strain in place of thecopper catalyst to promote a [3+2] azide-alkyne cycloaddition reaction(Id.) For example, cyclooctyne is an 8-carbon ring structure comprisingan internal alkyne bond. The closed ring structure induces a substantialbond angle deformation of the acetylene, which is highly reactive withazide groups to form a triazole. Thus, cyclooctyne derivatives may beused for copper-free click reactions (Id.)

Another type of copper-free click reaction was reported by Ning et al.(2010, Angew Chem Int Ed 49:3065-68), involving strain-promotedalkyne-nitrone cycloaddition. To address the slow rate of the originalcyclooctyne reaction, electron-withdrawing groups are attached adjacentto the triple bond (Id.) Examples of such substituted cyclooctynesinclude difluorinated cyclooctynes, 4-dibenzocyclooctynol andazacyclooctyne (Id.) An alternative copper-free reaction involvedstrain-promoted akyne-nitrone cycloaddition to give N-alkylatedisoxazolines (Id.) The reaction was reported to have exceptionally fastreaction kinetics and was used in a one-pot three-step protocol forsite-specific modification of peptides and proteins (Id.) Nitrones wereprepared by the condensation of appropriate aldehydes withN-methylhydroxylamine and the cycloaddition reaction took place in amixture of acetonitrile and water (Id.) These and other known clickchemistry reactions may be used to attach therapeutic agents toantibodies in vitro.

The specificity of the click chemistry reaction may be used as asubstitute for the antibody-hapten binding interaction used inpretargeting with bispecific antibodies. In this alternative embodiment,the specific reactivity of e.g., cyclooctyne moieties for azide moietiesor alkyne moieties for nitrone moieties may be used in an in vivocycloaddition reaction. An antibody, antibody fragment or antibody-basedcomplex is activated by incorporation of a substituted cyclooctyne, anazide or a nitrone moiety. A targetable construct is labeled with one ormore diagnostic or therapeutic agents and a complementary reactivemoiety. I.e., where the antibody comprises a cyclooctyne, the targetableconstruct will comprise an azide; where the antibody comprises anitrone, the targetable construct will comprise an alkyne, etc. Theactivated antibody or fragment is administered to a subject and allowedto localize to a targeted cell, tissue or pathogen, as disclosed forpretargeting protocols. The reactive labeled targetable construct isthen administered. Because the cyclooctyne, nitrone or azide on thetargetable construct is unreactive with endogenous biomolecules andhighly reactive with the complementary moiety on the antibody, thespecificity of the binding interaction results in the highly specificbinding of the targetable construct to the tissue-localized antibody.

Therapeutic Agents

A wide variety of therapeutic reagents can be administered concurrentlyor sequentially with the subject anti-CD22 antibodies or antibodycombinations. For example, drugs, toxins, oligonucleotides,immunomodulators, cytokine or chemokine inhibitors, proapoptotic agents,tyrosine kinase inhibitors, sphingosine inhibitors, hormones, hormoneantagonists, enzymes, enzyme inhibitors, radionuclides, angiogenesisinhibitors, other antibodies or fragments thereof, etc. The therapeuticagents recited here are those agents that also are useful foradministration separately with an antibody or fragment thereof asdescribed above. Therapeutic agents include, for example, cytotoxicagents such as vinca alkaloids, anthracyclines, gemcitabine,epipodophyllotoxins, taxanes, antimetabolites, alkylating agents,antibiotics, SN-38, COX-2 inhibitors, antimitotics, anti-angiogenic andpro-apoptotic agents, particularly doxorubicin, methotrexate, taxol,CPT-11, camptothecins, proteosome inhibitors, mTOR inhibitors, HDACinhibitors, tyrosine kinase inhibitors, and others.

Other useful cytotoxic agents include nitrogen mustards, alkylsulfonates, nitrosoureas, triazenes, folic acid analogs, COX-2inhibitors, antimetabolites, pyrimidine analogs, purine analogs,platinum coordination complexes, mTOR inhibitors, tyrosine kinaseinhibitors, proteosome inhibitors, HDAC inhibitors, camptothecins,hormones, and the like. Suitable cytotoxic agents are described inREMINGTON′S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co.1995), and in GOODMAN AND GILMAN′S THE PHARMACOLOGICAL BASIS OFTHERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as well asrevised editions of these publications.

In a preferred embodiment, conjugates of camptothecins and relatedcompounds, such as SN-38, may be conjugated to an antibody, for exampleas disclosed in U.S. Pat. No. 7,591,994, the Examples section of whichis incorporated herein by reference.

The therapeutic agent may be selected from the group consisting ofaplidin, azaribine, anastrozole, azacytidine, bleomycin, bortezomib,bryostatin-1, busulfan, calicheamycin, camptothecin,10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin,irinotecan (CPT-11), SN-38, carboplatin, cladribine, cyclophosphamide,cytarabine, dacarbazine, docetaxel, dactinomycin, daunomycinglucuronide, daunorubicin, dexamethasone, diethylstilbestrol,doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, ethinylestradiol, estramustine, etoposide, etoposide glucuronide, etoposidephosphate, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO),fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine,hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide,L-asparaginase, leucovorin, lomustine, mechlorethamine,medroprogesterone acetate, egestrol acetate, melphalan, mercaptopurine,6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin,mitotane, phenyl butyrate, prednisone, procarbazine, paclitaxel,pentostatin, PSI-341, semustine streptozocin, tamoxifen, taxanes, taxol,testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, velcade, vinblastine, vinorelbine,vincristine, ricin, abrin, ribonuclease, onconase, rapLR1, DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.

A toxin can be of animal, plant or microbial origin. A toxin, such asPseudomonas exotoxin, may also be complexed to or form the therapeuticagent portion of an immunoconjugate. Other toxins include ricin, abrin,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, onconase, gelonin, diphtheria toxin, Pseudomonasexotoxin, and Pseudomonas endotoxin. See, for example, Pastan et al.,Cell 47:641 (1986), Goldenberg, C A—A Cancer Journal for Clinicians44:43 (1994), Sharkey and Goldenberg, C A—A Cancer Journal forClinicians 56:226 (2006). Additional toxins suitable for use are knownto those of skill in the art and are disclosed in U.S. Pat. No.6,077,499, the Examples section of which is incorporated herein byreference.

The therapeutic agent may be an enzyme selected from the groupconsisting of malate dehydrogenase, staphylococcal nuclease,delta-V-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

As used herein, the term “immunomodulator” includes cytokines,lymphokines, monokines, stem cell growth factors, lymphotoxins,hematopoietic factors, colony stimulating factors (CSF), interferons(IFN), parathyroid hormone, thyroxine, insulin, proinsulin, relaxin,prorelaxin, follicle stimulating hormone (FSH), thyroid stimulatinghormone (TSH), luteinizing hormone (LH), hepatic growth factor,prostaglandin, fibroblast growth factor, prolactin, placental lactogen,OB protein, transforming growth factor (TGF), TGF-a, TGF-J3,insulin-like growth factor (IGF), erythropoietin, thrombopoietin, tumornecrosis factor (TNF), TNF-α, TNF-β, mullerian-inhibiting substance,mouse gonadotropin-associated peptide, inhibin, activin, vascularendothelial growth factor, integrin, interleukin (IL),granulocyte-colony stimulating factor (G-CSF), granulocytemacrophage-colony stimulating factor (GM-CSF), interferon-α,interferon-, interferon-, S1 factor, IL-1, IL-lcc, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18 IL-21, IL-23, IL-25, kit-ligand, FLT-3, angiostatin,thrombospondin, endostatin, and the like.

Exemplary anti-angiogenic agents may include angiostatin, endostatin,vasculostatin, canstatin, maspin, anti-VEGF binding molecules,anti-placental growth factor binding molecules, or anti-vascular growthfactor binding molecules.

In certain embodiments, the antibody or complex may comprise one or morechelating moieties, such as NOTA, DOTA, DTPA, TETA, Tscg-Cys, orTsca-Cys. In certain embodiments, the chelating moiety may form acomplex with a therapeutic or diagnostic cation, such as Group II, GroupIII, Group IV, Group V, transition, lanthanide or actinide metalcations, Tc, Re, Bi, Cu, As, Ag, Au, At, or Pb.

The antibody or fragment thereof may be administered as animmunoconjugate comprising one or more radioactive isotopes useful fortreating diseased tissue. Particularly useful therapeutic radionuclidesinclude, but are not limited to ¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu,⁶⁴Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm,¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe,⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au,¹⁹⁹Au, and ²¹¹Pb. The therapeutic radionuclide preferably has a decayenergy in the range of 20 to 6,000 keV, preferably in the ranges 60 to200 keV for an Auger emitter, 100-2,500 keV for a beta emitter and4,000-6,000 keV for an alpha emitter. Maximum decay energies of usefulbeta-particle-emitting nuclides are preferably 20-5,000 keV, morepreferably 100-4,000 keV and most preferably 500-2,500 keV. Alsopreferred are radionuclides that substantially decay with Auger-emittingparticles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109,In-111, Sb-119, 1-125, Ho-161, Os-189m and Ir-192. Decay energies ofuseful beta-particle-emitting nuclides are preferably <1,000 keV, morepreferably <100 keV, and most preferably <70 keV. Also preferred areradionuclides that substantially decay with generation ofalpha-particles. Such radionuclides include, but are not limited to:Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221,At-217, Bi-213 and Fm-255. Decay energies of usefulalpha-particle-emitting radionuclides are preferably 2,000-10,000 keV,more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.

Additional potential therapeutic radioisotopes include ¹¹C, ¹³N, ¹⁵O,⁷⁵Br, ¹⁹⁸Au, 224Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru,¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm,¹⁶⁸Tm, ¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co,⁵⁸Co, ⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

Interference RNA

In certain preferred embodiments the therapeutic agent may be a siRNA orinterference RNA species. The siRNA, interference RNA or therapeuticgene may be attached to a carrier moiety that is conjugated to anantibody or fragment thereof. A variety of carrier moieties for siRNAhave been reported and any such known carrier may be incorporated into atherapeutic antibody for use. Non-limiting examples of carriers includeprotamine (Rossi, 2005, Nat Biotech 23:682-84; Song et al., 2005, NatBiotech 23:709-17); dendrimers such as PAMAM dendrimers (Pan et al.,2007, Cancer Res. 67:8156-8163); polyethylenimine (Schiffelers et al.,2004, Nucl Acids Res 32:e149); polypropyleneimine (Taratula et al.,2009, J Control Release 140:284-93); polylysine (Inoue et al., 2008, JControl Release 126:59-66); histidine-containing reducible polycations(Stevenson et al., 2008, J Control Release 130:46-56); histone H1protein (Haberland et al., 2009, Mol Biol Rep 26:1083-93); cationiccomb-type copolymers (Sato et al., 2007, J Control Release 122:209-16);polymeric micelles (U.S. Patent Application Publ. No. 20100121043); andchitosan-thiamine pyrophosphate (Rojanarata et al., 2008, Pharm Res25:2807-14). The skilled artisan will realize that in general,polycationic proteins or polymers are of use as siRNA carriers. Theskilled artisan will further realize that siRNA carriers can also beused to carry other oligonucleotide or nucleic acid species, such asanti-sense oligonucleotides or short DNA genes.

Known siRNA species of potential use include those specific forIKK-gamma (U.S. Pat. No. 7,022,828); VEGF, Flt-1 and Flk-1/KDR (U.S.Pat. No. 7,148,342); Bc12 and EGFR (U.S. Pat. No. 7,541,453); CDC20(U.S. Pat. No. 7,550,572); transducin (beta)-like 3 (U.S. Pat. No.7,576,196); K-ras (U.S. Pat. No. 7,576,197); carbonic anhydrase II (U.S.Pat. No. 7,579,457); complement component 3 (U.S. Pat. No. 7,582,746);interleukin-1 receptor-associated kinase 4 (IRAK4) (U.S. Pat. No.7,592,443); survivin (U.S. Pat. No. 7,608,7070); superoxide dismutase 1(U.S. Pat. No. 7,632,938); MET proto-oncogene (U.S. Pat. No. 7,632,939);amyloid beta precursor protein (APP) (U.S. Pat. No. 7,635,771); IGF-1R(U.S. Pat. No. 7,638,621); ICAM1 (U.S. Pat. No. 7,642,349); complementfactor B (U.S. Pat. No. 7,696,344); p53 (7,781,575), and apolipoproteinB (7,795,421), the Examples section of each referenced patentincorporated herein by reference.

Additional siRNA species are available from known commercial sources,such as Sigma-Aldrich (St Louis, Mo.), Invitrogen (Carlsbad, Calif.),Santa Cruz Biotechnology (Santa Cruz, Calif.), Ambion (Austin, Tex.),Dharmacon (Thermo Scientific, Lafayette, Colo.), Promega (Madison,Wis.), Mirus Bio (Madison, Wis.) and Qiagen (Valencia, Calif.), amongmany others. Other publicly available sources of siRNA species includethe siRNAdb database at the Stockholm Bioinformatics Centre, theMIT/ICBP siRNA Database, the RNAi Consortium shRNA Library at the BroadInstitute, and the Probe database at NCBI. For example, there are 30,852siRNA species in the NCBI Probe database. The skilled artisan willrealize that for any gene of interest, either a siRNA species hasalready been designed, or one may readily be designed using publiclyavailable software tools. Any such siRNA species may be delivered usingthe subject antibodies, antibody fragments or antibody complexes.

Exemplary siRNA species known in the art are listed in Table 6. AlthoughsiRNA is delivered as a double-stranded molecule, for simplicity onlythe sense strand sequences are shown in Table 6.

TABLE 6 Exemplary siRNA Sequences SEQ ID Target Sequence NO VEGF R2AATGCGGCGGTGGTGACAGTA SEQ ID NO: 99 VEGF R2 AAGCTCAGCACACAGAAAGAC SEQ IDNO: 100 CXCR4 UAAAAUCUUCCUGCCCACCdT SEQ ID dT NO: 101 CXCR4GGAAGCUGUUGGCUGAAAAdT SEQ ID dT NO: 102 PPARC1 AAGACCAGCCUCUUUGCCCAGSEQ ID NO: 103 Dynamin 2 GGACCAGGCAGAAAACGAG SEQ ID NO: 104 CateninCUAUCAGGAUGACGCGG SEQ ID NO: 105 E1A  UGACACAGGCAGGCUUGACUU SEQ IDbinding  NO: 106 protein Plasminogen  GGTGAAGAAGGGCGTCCAA SEQ IDactivator NO: 107 K-ras GATCCGTTGGAGCTGTTGGCG SEQ IDTAGTTCAAGAGACTCGCCAAC NO: 108 AGCTCCAACTTTTGGAAA Sortilin 1AGGTGGTGTTAACAGCAGAG SEQ ID NO: 109 Apolipoprotein AAGGTGGAGCAAGCGGTGGAGSEQ ID E NO: 110 Apolipoprotein AAGGAGTTGAAGGCCGACAAA SEQ ID E NO: 111Bcl-X UAUGGAGCUGCAGAGGAUGdT SEQ ID dT NO: 112 Raf-1TTTGAATATCTGTGCTGAGAA SEQ ID CACAGTTCTCAGCACAGATAT NO: 113 TCTTTTTHeat shock AATGAGAAAAGCAAAAGGTGC SEQ ID transcription  CCTGTCTC NO: 114factor 2 IGFBP3 AAUCAUCAUCAAGAAAGGGCA SEQ ID NO: 115 ThioredoxinAUGACUGUCAGGAUGUUGCdT SEQ ID dT NO: 116 CD44 GAACGAAUCCUGAAGACAUCUSEQ ID NO: 117 MMP14 AAGCCTGGCTACAGCAATATG SEQ ID CCTGTCTC NO: 118MAPKAPK2 UGACCAUCACCGAGUUUAUdT SEQ ID dT NO: 119 FGFR1AAGTCGGACGCAACAGAGAAA SEQ ID NO: 120 ERBB2 CUACCUUUCUACGGACGUGdT SEQ IDdT NO: 121 BCL2L1 CTGCCTAAGGCGGATTTGAAT SEQ ID NO: 122 ABL1TTAUUCCUUCUUCGGGAAGUC SEQ ID NO: 123 CEACAM1 AACCTTCTGGAACCCGCCCACSEQ ID NO: 124 CD9 GAGCATCTTCGAGCAAGAA SEQ ID NO: 125 CD151CATGTGGCACCGTTTGCCT SEQ ID NO: 126 Caspase 8 AACTACCAGAAAGGTATACCTSEQ ID NO: 127 BRCA1 UCACAGUGUCCUUUAUGUAdT SEQ ID dT NO: 128 p53GCAUGAACCGGAGGCCCAUTT SEQ ID NO: 129 CEACAM6 CCGGACAGTTCCATGTATA SEQ IDNO: 130

The skilled artisan will realize that Table 6 represents a very smallsampling of the total number of siRNA species known in the art, and thatany such known siRNA may be utilized in the claimed methods andcompositions.

Immunotoxins Comprising Ranpirnase (Rap)

Ribonucleases, in particular, Rap (Lee, Exp Opin Biol Ther 2008;8:813-27) and its more basic variant, amphinase (Ardelt et al., CurrPharm Biotechnol 2008:9:215-25), are potential anti-tumor agents (Leeand Raines, Biodrugs 2008; 22:53-8). Rap is a single-chain ribonucleaseof 104 amino acids originally isolated from the oocytes of Rana pipiens.Rap exhibits cytostatic and cytotoxic effects on a variety of tumor celllines in vitro, as well as antitumor activity in vivo. The amphibianribonuclease enters cells via receptor-mediated endocytosis and onceinternalized into the cytosol, selectively degrades tRNA, resulting ininhibition of protein synthesis and induction of apoptosis.

Rap has completed a randomized Phase IIIb clinical trial, which comparedthe effectiveness of Rap plus doxorubicin with that of doxorubicin alonein patients with unresectable malignant mesothelioma, with the interimanalysis showing that the MST for the combination was 12 months, whilethat of the monotherapy was 10 months (Mutti and Gaudino, Oncol Rev2008; 2:61-5). Rap can be administered repeatedly to patients without anuntoward immune response, with reversible renal toxicity reported to bedose-limiting (Mikulski et al., J Clin Oncol 2002; 20:274-81; Int JOncol 1993; 3:57-64).

Conjugation or fusion of Rap to a tumor-targeting antibody or antibodyfragment is a promising approach to enhance its potency, as firstdemonstrated for LL2-onconase (Newton et al., Blood 2001; 97:528-35), achemical conjugate comprising Rap and a murine anti-CD22 monoclonalantibody (MAb), and subsequently for 2L-Rap-hLL1-4P, a fusion proteincomprising Rap and a humanized anti-CD74 MAb (Stein et al., Blood 2004;104:3705-11).

The method used to generate 2L-Rap-hLL1-4P allowed us to develop aseries of structurally similar immunotoxins, referred to in general as2L-Rap-X, all of which consist of two Rap molecules, each connected viaa flexible linker to the N-terminus of one L chain of an antibody ofinterest (X). We have also generated another series of immunotoxins ofthe same design, referred to as 2LRap(Q)-X, by substituting Rap with itsnon-glycosylation form of Rap, designated as Rap(Q) to denote that thepotential glycosylation site at Asn69 is changed to Gln (or Q, singleletter code). For both series, we made the IgG as either IgG1(1) orIgG4(4), and to prevent the formation of IgG4 half molecules (Aalberseand Schuurman, Immunology 2002; 105:9-19), we converted the serineresidue in the hinge region (S228) of IgG4 to proline (4P). Apyroglutamate residue at the N-terminus of Rap is required for the RNaseto be fully functional (Liao et al., Nucleic Acids Res 2003;31:5247-55).

The skilled artisan will recognize that the cytotoxic RNase moietiessuitable for use in the present invention include polypeptides having anative ranpirnase structure and all enzymatically active variantsthereof. These molecules advantageously have an N-terminal pyroglutamicacid resides that appears essential for RNase activity and are notsubstantially inhibited by mammalian RNase inhibitors. Nucleic acid thatencodes a native cytotoxic RNase may be prepared by cloning andrestriction of appropriate sequences, or using DNA amplification withpolymerase chain reaction (PCR). The amino acid sequence of Rana pipiensranpirnase can be obtained from Ardelt et al., J. Biol. Chem., 256: 245(1991), and cDNA sequences encoding native ranpirnase, or aconservatively modified variation thereof, can be gene-synthesized bymethods similar to the en bloc V-gene assembly method used in hLL2humanization. (Leung et al., Mol. Immunol., 32: 1413, 1995). Methods ofmaking cytotoxic RNase variants are known in the art and are within theskill of the routineer.

As described in the Examples below, Rap conjugates of targetingantibodies may be made using the DNL technology. The DNL Rap-antibodyconstructs show potent cytotoxic activity that can be targeted todisease-associated cells.

Diagnostic Agents

In various embodiments, the antibodies, antibody fragments or antibodycomplexes may be conjugated to, or may bind a targetable constructcomprising one or more diagnostic agents. Diagnostic agents arepreferably selected from the group consisting of a radionuclide, aradiological contrast agent, a paramagnetic ion, a metal, a fluorescentlabel, a chemiluminescent label, an ultrasound contrast agent and aphotoactive agent. Such diagnostic agents are well known and any suchknown diagnostic agent may be used. Non-limiting examples of diagnosticagents may include a radionuclide such as ¹⁸F, ⁵²Fe, ¹¹⁰In, ¹¹¹In,¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr,^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ³²P,¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^(52m)Mn, ⁵⁵Co, ⁷²AS, ⁷⁵Br, ⁷⁶Br,^(82m)Rb, ⁸³Sr, or other gamma-, beta-, or positron-emitters.

Paramagnetic ions of use may include chromium (III), manganese (II),iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) or erbium (III). Metalcontrast agents may include lanthanum (III), gold (III), lead (II) orbismuth (III).

Ultrasound contrast agents may comprise liposomes, such as gas filledliposomes. Radiopaque diagnostic agents may be selected from compounds,barium compounds, gallium compounds, and thallium compounds. A widevariety of fluorescent labels are known in the art, including but notlimited to fluorescein isothiocyanate, rhodamine, phycoerytherin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.Chemiluminescent labels of use may include luminol, isoluminol, anaromatic acridinium ester, an imidazole, an acridinium salt or anoxalate ester.

Methods of Therapeutic Treatment

The claimed methods and compositions are of use for treating diseasestates, such as B-cell lymphomas or leukemias, autoimmune disease orimmune system dysfunction (e.g., graft-versus-host disease). The methodsmay comprise administering a therapeutically effective amount of ananti-CD22 antibody or fragment thereof or immunoconjugate, either aloneor in combination with one or more other therapeutic agents,administered either concurrently or sequentially. In preferredembodiments, as described in the Examples below, the anti-CD22 antibodyor fragment thereof may be administered in the form of a DNL complex incombination with one or more other therapeutic agents, such as a secondantibody or fragment thereof.

Multimodal therapies may include therapy with other antibodies, such asantibodies against CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22,CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80,CD126, CD138, B7, HM1.24, HLA-DR, an angiogenesis factor, tenascin,VEGF, P1GF, ED-B fibronectin, an oncogene, an oncogene product, NCA66a-d, necrosis antigens, Ii, IL-2, T101, TAC, MUC-1, TRAIL-R1 (DR4) orTRAIL-R2 (DR5) in the form of naked antibodies, fusion proteins, or asimmunoconjugates. Various antibodies of use are known to those of skillin the art. See, for example, Ghetie et al., Cancer Res. 48:2610 (1988);Hekman et al., Cancer Immunol. Immunother. 32:364 (1991); Longo, Curr.Opin. Oncol. 8:353 (1996), U.S. Pat. Nos. 5,798,554; 6,187,287;6,306,393; 6,676,924; 7,109,304; 7,151,164; 7,230,084; 7,230,085;7,238,785; 7,238,786; 7,282,567; 7,300,655; 7,312,318; 7,612,180;7,501,498; the Examples section of each of which is incorporated hereinby reference.

In another form of multimodal therapy, subjects may receive therapeuticanti-CD22 antibodies or antibody combinations in conjunction withstandard chemotherapy. For example, “CVB” (1.5 g/m² cyclophosphamide,200-400 mg/m² etoposide, and 150-200 mg/m² carmustine) is a regimen usedto treat non-Hodgkin's lymphoma. Patti et al., Eur. J. Haematol. 51: 18(1993). Other suitable combination chemotherapeutic regimens arewell-known to those of skill in the art. See, for example, Freedman etal., “Non-Hodgkin's Lymphomas,” in CANCER MEDICINE, VOLUME 2, 3rdEdition, Holland et al. (eds.), pages 2028-2068 (Lea & Febiger 1993). Asan illustration, first generation chemotherapeutic regimens fortreatment of intermediate-grade non-Hodgkin's lymphoma (NHL) includeC-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone) andCHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). Auseful second generation chemotherapeutic regimen is m-BACOD(methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine,dexamethasone and leucovorin), while a suitable third generation regimenis MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine,prednisone, bleomycin and leucovorin). Additional useful drugs includephenyl butyrate, bendamustine, and bryostatin-1.

Therapeutic antibodies or complexes, such as DNL complexes, can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby the therapeutic antibody complex is combined in amixture with a pharmaceutically suitable excipient. Sterilephosphate-buffered saline is one example of a pharmaceutically suitableexcipient. Other suitable excipients are well-known to those in the art.See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUGDELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),REMINGTON′S PHARMACEUTICAL SCIENCES, 18th Edition (Mack PublishingCompany 1990), and revised editions thereof.

The therapeutic antibody complex can be formulated for intravenousadministration via, for example, bolus injection or continuous infusion.Preferably, the therapeutic antibody complex is infused over a period ofless than about 4 hours, and more preferably, over a period of less thanabout 3 hours. For example, the first 25-50 mg could be infused within30 minutes, preferably even 15 min, and the remainder infused over thenext 2-3 hrs. Formulations for injection can be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions can take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The therapeutic antibody complex may also be administered to a mammalsubcutaneously or even by other parenteral routes. Moreover, theadministration may be by continuous infusion or by single or multipleboluses. In most preferred embodiments, the therapeutic antibody orcombination is administered subcutaneously in a volume of 1, 2 or 3 mland at a concentration of at least 80 mg/ml, at least 100 mg/ml, atleast 125 mg/ml, at least 150 mg/ml, at least 200 mg/ml, at least 250mg/ml or at least 300 mg/ml. Methods of antibody concentration andsubcutaneous formulations are disclosed in provisional U.S. Patent No.61/509,850, filed Jul. 20, 2011, the Examples section of which (fromparagraph 0133, page 48 to paragraph 0195, page 64) is incorporatedherein by reference.

More generally, the dosage of an administered therapeutic antibodycomplex for humans will vary depending upon such factors as thepatient's age, weight, height, sex, general medical condition andprevious medical history. It may be desirable to provide the recipientwith a dosage of therapeutic antibody complex that is in the range offrom about 1 mg/kg to 25 mg/kg as a single intravenous infusion,although a lower or higher dosage also may be administered ascircumstances dictate. A dosage of 1-20 mg/kg for a 70 kg patient, forexample, is 70-1,400 mg, or 41-824 mg/m² for a 1.7-m patient. The dosagemay be repeated as needed, for example, once per week for 4-10 weeks,once per week for 8 weeks, or once per week for 4 weeks. It may also begiven less frequently, such as every other week for several months, ormonthly or quarterly for many months, as needed in a maintenancetherapy.

Alternatively, a therapeutic antibody complex may be administered as onedosage every 2 or 3 weeks, repeated for a total of at least 3 dosages.Or, the therapeutic antibody complex may be administered twice per weekfor 4-6 weeks. If the dosage is lowered to approximately 200-300 mg/m²(340 mg per dosage for a 1.7-m patient, or 4.9 mg/kg for a 70 kgpatient), it may be administered once or even twice weekly for 4 to 10weeks. Alternatively, the dosage schedule may be decreased, namely every2 or 3 weeks for 2-3 months. It has been determined, however, that evenhigher doses, such as 20 mg/kg once weekly or once every 2-3 weeks canbe administered by slow i.v. infusion, for repeated dosing cycles. Thedosing schedule can optionally be repeated at other intervals and dosagemay be given through various parenteral routes, with appropriateadjustment of the dose and schedule.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic immunoconjugate or naked antibody.Control release preparations can be prepared through the use of polymersto complex or adsorb the antibody. For example, biocompatible polymersinclude matrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid.Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of release ofan antibody from such a matrix depends upon the molecular weight of theantibody, the amount of antibody within the matrix, and the size ofdispersed particles. Saltzman et al., Biophys. J. 55: 163 (1989);Sherwood et al., supra. Other solid dosage forms are described in Anselet al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON′SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

Cancer Therapy

In preferred embodiments, the anti-CD22 antibodies, combinations orcomplexes are of use for therapy of cancer. Examples of cancers include,but are not limited to, carcinoma, lymphoma, glioblastoma, melanoma,sarcoma, and leukemia, myeloma, or lymphoid malignancies. Moreparticular examples of such cancers are noted below and include:squamous cell cancer (e.g., epithelial squamous cell cancer), Ewingsarcoma, Wilms tumor, astrocytomas, lung cancer including small-celllung cancer, non-small cell lung cancer, adenocarcinoma of the lung andsquamous carcinoma of the lung, cancer of the peritoneum, hepatocellularcancer, gastric or stomach cancer including gastrointestinal cancer,pancreatic cancer, glioblastoma multiforme, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, hepatocellularcarcinoma, neuroendocrine tumors, medullary thyroid cancer,differentiated thyroid carcinoma, breast cancer, ovarian cancer, coloncancer, rectal cancer, endometrial cancer or uterine carcinoma, salivarygland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer,anal carcinoma, penile carcinoma, as well as head-and-neck cancer. Theterm “cancer” includes primary malignant cells or tumors (e.g., thosewhose cells have not migrated to sites in the subject's body other thanthe site of the original malignancy or tumor) and secondary malignantcells or tumors (e.g., those arising from metastasis, the migration ofmalignant cells or tumor cells to secondary sites that are differentfrom the site of the original tumor).

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In preferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Therapy of Autoimmune Disease

The subject anti-CD22 antibodies, combinations or complexes thereof canbe used to treat immune dysregulation disease and related autoimmunediseases. Immune diseases may include acute immune thrombocytopenia,Addison's disease, adult respiratory distress syndrome (ARDS),agranulocytosis, allergic conditions, allergic encephalomyelitis,allergic neuritis, amyotrophic lateral sclerosis (ALS), ankylosingspondylitis, antigen-antibody complex mediated diseases, anti-glomerularbasement membrane disease, anti-phospholipid antibody syndrome, aplasticanemia, arthritis, asthma, atherosclerosis, autoimmune disease of thetestis and ovary, autoimmune endocrine diseases, autoimmune myocarditis,autoimmune neutropenia, autoimmune polyendocrinopathies, autoimmunepolyglandular syndromes (or polyglandular endocrinopathy syndromes),autoimmune thrombocytopenia, Bechet disease, Berger's disease (IgAnephropathy), bronchiolitis obliterans (non-transplant), bullouspemphigoid, pemphigus vulgaris, Castleman's syndrome, Celiac sprue(gluten enteropathy), central nervous system (CNS) inflammatorydisorders, chronic active hepatitis, chronic immune thrombocytopeniadermatomyositis, colitis, conditions involving infiltration of T cellsand chronic inflammatory responses, coronary artery disease, Crohn'sdisease, cryoglobulinemia, dermatitis, dermatomyositis, diabetesmellitus, diseases involving leukocyte diapedesis, eczema, encephalitis,erythema multiforme, erythema nodosum, Factor VIII deficiency, fibrosingalveolitis, giant cell arteritis, glomerulonephritis, Goodpasture'ssyndrome, graft versus host disease (GVHD), granulomatosis, Grave'sdisease, Guillain-Barre Syndrome, Hashimoto's thyroiditis, hemophilia A,Henoch-Schonlein purpura, idiopathic hypothyroidism, immunethrombocytopenia (ITP), IgA nephropathy, IgA nephropathy, IgM mediatedneuropathy, immune complex nephritis, immune hemolytic anemia includingautoimmune hemolytic anemia (AIHA), immune responses associated withacute and delayed hypersensitivity mediated by cytokines andT-lymphocytes, immune-mediated thrombocytopenias, juvenile onsetdiabetes, juvenile rheumatoid arthritis, Lambert-Eaton MyasthenicSyndrome, large vessel vasculitis, leukocyte adhesion deficiency,leukopenia, lupus nephritis, lymphoid interstitial pneumonitis (HIV),medium vessel vasculitis, membranous nephropathy, meningitis, multipleorgan injury syndrome, multiple sclerosis, myasthenia gravis,osteoarthritis, pancytopenia, pemphigoid bullous, pemphigus vulgaris,pernicious anemia, polyarteritis nodosa, polychondritis, polyglandularsyndromes, polymyalgia, polymyositis, post-streptococcal nephritis,primary biliary cirrhosis, primary hypothyroidism, psoriasis, psoriaticarthritis, pure red cell aplasia (PRCA), rapidly progressiveglomerulonephritis, Reiter's disease, respiratory distress syndrome,responses associated with inflammatory bowel disease, Reynaud'ssyndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis,scleroderma, Sjogren's syndrome, solid organ transplant rejection,Stevens-Johnson syndrome, stiff-man syndrome, subacute thyroiditis,Sydenham's chorea, systemic lupus erythematosus (SLE), systemicscleroderma and sclerosis, tabes dorsalis, Takayasu's arteritis,thromboangitis obliterans, thrombotic thrombocytopenic purpura (TTP),thyrotoxicosis, toxic epidermal necrolysis, tuberculosis, Type Idiabetes, ulcerative colitis, uveitis, vasculitis (including ANCA) andWegener's granulomatosis.

Type-1 and Type-2 diabetes may be treated using known antibodies againstB-cell antigens, such as CD22 (epratuzumab), CD74 (milatuzumab), CD19(hA19), CD20 (veltuzumab) or HLA-DR (hL243) (see, e.g., Winer et al.,2011, Nature Med 17:610-18). Anti-CD3 antibodies also have been proposedfor therapy of type 1 diabetes (Cernea et al., 2010, Diabetes Metab Rev26:602-05).

Kits

Various embodiments may concern kits containing anti-CD22 antibodies,antibody combinations and/or antibody constructs and/or othercomponents. Such components may include a targetable construct. Inalternative embodiments it is contemplated that a targetable constructmay be attached to one or more different therapeutic and/or diagnosticagents.

If the composition containing components for administration is notformulated for delivery via the alimentary canal, such as by oraldelivery, a device capable of delivering the kit components through someother route may be included. One type of device, for applications suchas parenteral delivery, is a syringe that is used to inject thecomposition into the body of a subject. Inhalation devices may also beused for certain applications.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

EXAMPLES

Various embodiments of the present invention are illustrated by thefollowing examples, without limiting the scope thereof.

General Procedures

Abbreviations used below are: DCC, dicyclohexylcarbodiimide; NHS,N-hydroxysuccinimide, DMAP, 4-dimethylaminopyridine; EEDQ,2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; MMT, monomethoxytrityl;PABOH, p-aminobenzyl alcohol; PEG, polyethylene glycol; SMCC,succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; TBAF,tetrabutylammonium fluoride; TBDMS, tert-butyldimethylsilyl chloride.

Chloroformates of hydroxy compounds in the following examples wereprepared using triphosgene and DMAP according to the procedure describedin Moon et al. (J. Medicinal Chem. 51:6916-6926, 2008). Extractivework-up refers to extraction with chloroform, dichloromethane or ethylacetate, and washing optionally with saturated bicarbonate, water, andwith saturated sodium chloride. Flash chromatography was done using230-400 mesh silica gel and a methanol-dichloromethane gradient, usingup to 15% v/v methanol-dichloromethane, unless otherwise stated. Reversephase HPLC was performed by Method A using a 7.8×300 mm C18 HPLC column,fitted with a precolumn filter, and using a solvent gradient of 100%solvent A to 100% solvent B in 10 minutes at a flow rate of 3 mL perminute and maintaining at 100% solvent B at a flow rate of 4.5 mL perminute for 5 or 10 minutes; or by Method B using a 4.6×30 mm C18, 2.5μm, column, fitted with a precolumn filter, using the solvent gradientof 100% solvent A to 100% of solvent B at a flow rate of 1.5 mL perminutes for 4 min and 100% of solvent B at a flow rate of 2 mL perminutes for 1 minutes. Solvent A was 0.3% aqueous ammonium acetate, pH4.46 while solvent B was 9:1 acetonitrile-aqueous ammonium acetate(0.3%), pH 4.46. HPLC was monitored by a dual in-line absorbancedetector set at 360 nm and 254 nm.

Example 1 Preparation of CL6-SN-38

An exemplary synthetic protocol for CL6-SN-38 is presented in Scheme 1.Commercially availableO-(2-azidoethyl)-O′—(N-diglycolyl-2-aminoethyl)heptaethyleneglycol(‘PEG-N₃’; 227 mg) was activated with DCC (100 mg), NHS (56 mg), and acatalytic amount of DMAP in 10 mL of dichloromethane for 10 min. To thismixture was added L-valinol (46.3 mg), and the reaction mixture wasstirred for 1 h at ambient temperature. Filtration, followed by solventremoval and flash chromatography yielded 214 mg of clear oily material.This intermediate (160 mg) was reacted with10-O-BOC-SN-38-20-O-chloroformate, the latter generated from10-O-BOC-SN-38 (123 mg) using triphosgene and DMAP. The couplingreaction was done in 4 mL of dichloromethane for 10 min, and thereaction mixture was purified by flash chromatography to obtain 130 mg(45% yield) of product as foamy material. HPLC: t_(R) 11.80 min;electrospray mass spectrum: M+Na: m/z 1181.

A maleimide-containing acetylenic reagent,4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide, requiredfor click cycloaddition, was prepared by reacting 0.107 g of SMCC and0.021 mL of propargylamine (0.018 g; 1.01 equiv.) in dichloromethaneusing 1.1 equiv. of diisopropylethylamine. After 1 h, the solvent wasremoved and the product was purified by flash chromatography to obtain83 mg of the product (colorless powder). Electrospray mass spectrumshowed peaks at m/e 275 (M+H) and a base peak at m/e 192 in the positiveion mode, consistent with the structure calculated for C₁₅H₁₈N₂O₃:275.1390 (M+H), found: 275.1394 (exact mass).

The azido intermediate (126 mg) described above was dissolved in DMSO(1.5 mL) and water (0.4 mL), and reacted with 60 mg of4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide and 15 mgof cuprous bromide and stirred for 30 min at ambient temperature. Flashchromatography, after work up of the reaction mixture, furnished 116 mg(75% yield) of the cycloaddition product. HPLC: t_(R) 11.20 min;electrospray mass spectrum: M+H and M+Na at m/z 1433 and 1456,respectively. Finally, deprotection with a mixture of TFA (5 mL),dichloromethane (1 mL), anisole (0.1 mL) and water (0.05 mL), followedby precipitation with ether and subsequent flash chromatography yieldedthe product, CL6-SN-38, as a gummy material. HPLC: t_(R) 9.98 min;electrospray mass spectrum: M+H and M−H (negative ion mode) at m/z 1333and 1356, respectively.

Example 2 Preparation of CL7-SN-38

The synthesis of CL7-SN-38 is schematically shown in Scheme 2. L-Valinol(40 mg) was reacted with commercially available Fmoc-Lys(MMT)-OH (253mg) and EEDQ (107 mg) in 10 mL of anhydrous dichloromethane at ambienttemperature, under argon, for 3 h. Extractive work up followed by flashchromatography furnished the product Fmoc-Lys(MMT)-valinol as a paleyellow liquid (200 mg; 70% yield). HPLC: t_(R) 14.38 min; electrospraymass spectrum: M+H: m/z 727. This intermediate (200 mg) was deprotectedwith diethylamine (10 mL), and the product (135 mg) was obtained in ˜90%purity after flash chromatography. HPLC: t_(R) 10.91 min; electrospraymass spectrum: M+Na at m/z 527. This product (135 mg) was coupled withthe commercially availableO-(2-azidoethyl)-O′—(N-diglycolyl-2-aminoethyl)heptaethyleneglycol('PEG-N₃′; 150 mg, 1.1 equiv.) in presence of EEDQ (72 mg, 1.1 equiv.)in 10 mL of dichloromethane, and stirred overnight at ambienttemperature. The crude material was purified by flash chromatography toobtain 240 mg of the purified product as a light yellow oil (˜87%yield). HPLC: t_(R) 11.55 min; electrospray mass spectrum: M+H and M+Naat m/z 1041 and 1063, respectively.

This intermediate (240 mg) was reacted with10-O-TBDMS-SN-38-20-β-chloroformate, the latter generated from10-O-TBDMS-SN-38 (122 mg) using triphosgene and DMAP. The couplingreaction was done in 5 mL of dichloromethane for 10 min, and thereaction mixture was purified by flash chromatography to obtain 327 mgof product as pale yellow foam. Electrospray mass spectrum: M+H at m/z1574. The entire product was reacted with 0.25 mmol of TBAF in 10 mL ofdichloromethane for 5 min, and the reaction mixture was diluted to 100mL and washed with brine. Crude product (250 mg) was dissolved in DMSO(2 mL) and water (0.4 mL), and reacted with 114 mg of4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide (preparedas described in Example 1) and 30 mg of cuprous bromide and stirred for1 h at ambient temperature. Flash chromatography furnished 150 mg of thepenultimate intermediate. Finally, deprotection of the MMT group with amixture of TFA (0.5 mL) and anisole (0.05 mL) in dichloromethane (5 mL)for 3 min, followed by purification by flash chromatography yielded 69mg of CL7-SN-38 as a gummy material. HPLC: t_(R) 9.60 min; electrospraymass spectrum: M+H and M−H (negative ion mode) at m/z 1461 and 1459,respectively.

Example 3 Preparation of CL6-SN-38-10-O—CO₂Et

The CL6-SN-38 of Example 1 (55.4 mg) was dissolved in dichloromethane (5mL), and reacted with ethylchloroformate (13.1 mg; 11.5 μL) anddiisopropylethylamine (52.5 mg; 71 μL), and stirred for 20 min underargon. The reaction mixture was diluted with 100 mL of dichloromethane,and washed with 100 mL each of 0.1 M HCl, half saturated sodiumbicarbonate and brine, and dried. Flash chromatography, after solventremoval, furnished 59 mg of the title product. HPLC: t_(R) 10.74 min;exact mass: calc. 1404.6457 (M+H) and 1426.6276 (M+Na); found: 1404.6464(M+H) and 1426.6288 (M+Na).

Example 4 Preparation of CL7-SN-38-10-O—CO₂Et

The precursor of CL7-SN-38 of Example 2 (80 mg) was converted to the10-O-chloroformate using the procedure and purification as described inExample 3. Yield: 60 mg. HPLC: t_(R) 12.32 min; electrospray massspectrum: M+H and M−H (negative ion mode) at m/z 1806 and 1804,respectively. Deprotection of this material using dichloroacetic acidand anisole in dichloromethane gave CL7-SN-38-10-O—CO₂Et. HPLC: t_(R)10.37 min; electrospray mass spectrum: M+H at m/z 1534.

Example 5 Preparations of CL6-SN-38-10-O-COR and CL7-SN-38-10-O—COR

This Example shows that the 10-OH group of SN-38 is protected as acarbonate or an ester, instead of as ‘BOC’, such that the final productis ready for conjugation to antibodies without need for deprotecting the10-OH protecting group. This group is readily deprotected underphysiological pH conditions after in vivo administration of the proteinconjugate. In these conjugates, ‘R’ can be a substituted alkyl such as(CH₂)_(n)—N(CH₃)₂ where n is 2-10, or a simple alkyl such as(CH₂)_(n)—CH₃ where n is 0-10, or it can be an alkoxy moiety such as“CH₃—(CH₂)_(n)—O—” where n is 0-10, or a substituted alkoxy moiety suchas such as O—(CH₂)_(n)—N(CH₃)₂ where n is 2-10 and wherein the terminalamino group is optionally in the form of a quaternary salt for enhancedaqueous solubility, or “R₁₀-(CH₂—CH₂—O)_(n)—CH₂—CH₂—O—” where R₁ isethyl or methyl and n is an integer with values of 0-10. In the simplestversion of the latter category, R═“—O—(CH₂)₂—OCH₃”. These 10-hydroxyderivatives are readily prepared by treatment with the chloroformate ofthe chosen reagent, if the final derivative is to be a carbonate.Typically, the 10-hydroxy-containing camptothecin such as SN-38 istreated with a molar equivalent of the chloroformate indimethylformamide using triethylamine as the base. Under theseconditions, the 20-OH position is unaffected. For forming 10-O-esters,the acid chloride of the chosen reagent is used. Such derivatizationsare conveniently accomplished using advanced intermediates asillustrated for simple ethyl carbonates of Examples 3 and 4.

Example 6 Preparation of CL6-paclitaxel

Valinol is coupled to ‘PEG-N3’ of Scheme 1 according to the proceduredescribed in Example 1. The product is reacted with 0.4 molar equivalentof triphosgene, 3.1 molar equivalent of DMAP, in dichloromethane. After5 minutes, the chloroformate so formed is reacted with an equimolaramount of paclitaxel for 15 minutes at ambient temperature. The reactive2′-hydroxyl group of paclitaxel (the side chain secondary hydroxylgroup) reacts with the chloroformate of the cross-linker. The product isisolated by flash chromatography. This intermediate (0.1 mmol) isdissolved in DMSO (1.5 mL) and water (0.4 mL), and reacted with 60 mg of4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide (preparedas described in Example 1) and 15 mg of cuprous bromide and stirred for30 min at ambient temperature. Flash chromatography, after work up ofthe reaction mixture, furnishes the bifunctional paclitaxel, namelyCL6-paclitaxel.

Example 7 Preparation of CL7-paclitaxel

L-Valinol (40 mg) is reacted with commercially availableFmoc-Lys(MMT)-OH, and the product is then reacted withO-(2-azidoethyl)-O′—(N-diglycolyl-2-aminoethyl)heptaethyleneglycol(PEG-N₃′), as described in Example 2. The chloroformate of thisderivative is formed by the method of Example-6, and reacted with anequimolar amount of paclitaxel. The reactive 2′-hydroxyl group ofpaclitaxel (the side chain secondary hydroxyl group) reacts with thechloroformate of the cross-linker. Click cycloaddition, using4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide (preparedas described in Example 1) is then performed in a manner similar to thatdescribed in Example 6, and the product is finally treated withdichloroacetic acid and anisole to effect removal of the ‘MMT’ groupunder mild conditions. This process furnishes CL7-paclitaxel.

Example 8 Preparation of CL6-[morpholino doxorubicin]

Valinol is coupled to ‘PEG-N3’ of Scheme 1 according to the proceduredescribed in Example 1. The product is reacted with 0.4 molar equivalentof triphosgene, 3.1 molar equivalent of DMAP, in dichloromethane. After5 minutes, the chloroformate so formed is reacted with an equimolaramount of morpholino doxorubicin for 15 minutes at ambient temperature.The primary hydroxyl group of morpholino doxorubicin reacts with thechloroformate of the cross-linker. The product is isolated by flashchromatography. This intermediate (0.1 mmol) is dissolved in DMSO (1.5mL) and water (0.4 mL), and reacted with 60 mg of4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide (preparedas described in Example 1) and 15 mg of cuprous bromide and stirred for30 min at ambient temperature. Flash chromatography, after work up ofthe reaction mixture, furnishes the bifunctional paclitaxel, namelyCL6-[morpholino doxorubicin].

Example 9 Preparation of CL7-[morpholino doxorubicin]

L-Valinol (40 mg) is reacted with commercially availableFmoc-Lys(MMT)-OH, and the product is then reacted withO-(2-azidoethyl)-O′—(N-diglycolyl-2-aminoethyl)heptaethyleneglycol('PEG-N₃′), as described in Example 2. The chloroformate of thisderivative is formed by the method of Example-6, and reacted with anequimolar amount of morpholino doxorubicin. The primary hydroxyl groupof morpholino doxorubicin reacts with the chloroformate of thecross-linker. Click cycloaddition, using4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide (preparedas described in Example 1) is then performed in a manner similar to thatdescribed in Example 6, and the product is finally treated withdichloroacetic acid and anisole to effect removal of the ‘MMT’ groupunder mild conditions. This process furnishes CL7-[morpholinodoxorubicin].

Example 10 Preparation of CL2A-SN-38

An exemplary method for preparing CL2A-SN-38 is shown in Scheme 3. EEDQ(0.382 g) was added to a mixture of commercially availableFmoc-Lys(MMT)-OH (0.943 g), p-aminobenzyl alcohol (0.190 g) in methylenechloride (10 mL) at room temperature and stirred for 4 h. Extractivework up followed by flash chromatograph yielded 1.051 g of material aswhite foam. All HPLC analyses were performed by Method B as stated in‘General Procedures’. HPLC retention time was 3.53 min. Electrospraymass spectrum showed peaks at m/e 745.8 (M+H) and m/e 780.3 (M+Cl⁻),consistent with structure. This intermediate (0.93 g) was dissolved indiethylamine (10 mL) and stirred for 2 h. After solvent removal, theresidue was washed in hexane to obtain 0.6 g of the intermediate ((2) inScheme 3) as a colorless precipitate (91.6% pure by HPLC). HPLCretention time was 2.06 min. Electrospray mass spectrum showed peaks atm/e 523.8 (M+H), m/e 546.2 (M+Na) and m/e 522.5 (M−H). This crudeintermediate (0.565 g) was coupled with commercially available0-(2-azidoethyl)-O′—(N-diglycolyl-2-aminoethyl)heptaethyleneglycol(‘PEG-N3’, 0.627 g) using EEDQ in methylene chloride (10 mL). Solventremoval and flash chromatography yielded 0.99 g of the product ((3) inScheme 3; light yellow oil; 87% yield). HPLC retention time was 2.45min. Electrospray mass spectrum showed peaks at m/e 1061.3 (M+H), m/e1082.7 (M+Na) and m/e 1058.8(M−H), consistent with structure. Thisintermediate (0.92 g) was reacted with10-O-TBDMS-SN-38-20-O-chloroformate ((5) in Scheme 3) in methylenechloride (10 mL) for 10 min under argon. The mixture was purified byflash chromatography to obtain 0.944 g as a light yellow oil ((6) inScheme 3; yield=68%). HPLC retention time was 4.18 min. To thisintermediate (0.94 g) in methylene chloride (10 mL) was added a mixtureof TBAF (1M in THF, 0.885 mL) and acetic acid (0.085 mL) in methylenechloride (3 mL), then stirred for 10 min. The mixture was diluted withmethylene chloride (100 mL) and washed with 0.25 M sodium citrate andbrine. The solvent removal yielded 0.835 g of yellow oily product. HPLCretention time was 2.80 min (99% purity). Electrospray mass spectrumshowed peaks at m/e 1478 (M+H), m/e 1500.6 (M+Na), m/e 1476.5 (M−H), m/e1590.5 (M+TFA), consistent with structure.

This azido-derivatized SN-38 intermediate (0.803 g) was reacted with4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide (0.233 g)in methylene chloride (10 mL) in the presence of CuBr (0.0083 g), DMA(0.01 mL) and triphenylphosphine (0.015 g), for 18 h. Extractive workup, including washing with 0.1M EDTA (10 mL), and flash chromatographyyielded 0.891 g as yellow foam (yield=93%). HPLC retention time was 2.60min. The electrospray mass spectrum showed peaks at m/e 1753.3 (M+H),m/e 1751.6 (M−H), 1864.5 (M+TFA), consistent with structure. Finally,deprotection of the penultimate intermediate (0.22 g) with a mixture ofdichloroacetic acid (0.3 mL) and anisole (0.03 mL) in methylene chloride(3 mL), followed by precipitation with ether yielded 0.18 g (97% yield)of CL2A-SN-38; (7) in Scheme 3) as a light yellow powder. HPLC retentiontime was 1.88 min. Electrospray mass spectrum showed peaks at m/e 1480.7(M+H), 1478.5 (M−H), consistent with structure.

Example 11 Preparation of CL2E-SN-38

An exemplary method for preparing CL2E-SN-38 is shown in Scheme 4.N,N′-dimethylethylenediamine (3 mL) in methylene chloride (50 mL) wasreacted with monomethoxytrityl chloride (1.7 g). After 1 h of stirring,the solvent was removed under reduced pressure, and the crude productwas recovered by extractive work up (yellow oil; 2.13 g). All HPLCanalyses were performed by Method B as stated in ‘General Procedures’.HPLC retention time was 2.28 min. This intermediate ((1) in Scheme 4;0.93 g) was added in situ to activated SN-38, and the latter ((2) inScheme 4) was prepared by reacting SN-38 (0.3 g) withp-nitrophenylchloroformate (0.185 g) and DIEA (0.293 mL) in DMF for 1 h.After removing solvent, the residue was purified on deactivated silicagel to obtain 0.442 g as white solid. This intermediate (0.442 g) wasdeprotected with a mixture of trifluoroacetic acid (1 mL) and anisole(0.1 mL) in methylene chloride (5 mL), followed by precipitation withether to obtain 0.197 g of the product ((3) in Scheme 4) as white solid.

This intermediate (0.197 g) was coupled with activatedazide-containing-dipeptide incorporated-PEG-linker ((5) in Scheme 4),which activation was done by reacting PEG-linker ((4) in Scheme 4; 0.203g) with bis(4-nitrophenyl) carbonate (0.153 g) and DIEA (0.044 mL) inmethylene chloride (8 mL). Flash chromatography yielded 0.2 g ofazide-derivatized SN-38 intermediate product ((6) in Scheme 4) as aglassy solid. HPLC ret. time: 2.8 min. Electrospray mass spectrum showedpeaks at m/e 1740.5 (M+H), m/e 1762.9 (M+Na), m/e 1774.9 (M+Cl⁻),consistent with structure. This intermediate ((6) in Scheme 4; 0.2 g)was subjected to click cycloaddition with4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide (0.067 g)in methylene chloride in the presence of CuBr (0.007 g), DIEA (0.008 mL)and triphenylphosphine (0.012 g) for 18 h. Work up of the reactionmixture, which included treatment with 0.1M EDTA, followed by flashchromatography yielded 0.08 g of the penultimate intermediate as a lightyellow foam. HPLC: t_(R)=2.63 min. Electrospray mass spectrum showedpeaks at m/e 2035.9 (M+Na⁺), m/e 2047.9 (M+Cl⁻), consistent withstructure. Finally, deprotection of this intermediate (0.08 g) with amixture of trifluoroacetic acid (0.2 mL), anisole (0.12 mL) and water(0.06 mL) in methylene chloride (2 mL), followed by precipitation withether yielded 0.051 g of product, CL17-SN-38 (also referred to asCL2E-SN-38), as a light yellow powder (yield=69%). HPLC ret. time: 1.95min., ˜99% purity. Electrospray mass spectrum showed peaks at m/e 1741.1(M+H), 1775.5 (M+Cl⁻), consistent with structure.

Example 12 Conjugation of Bifunctional SN-38 Products to Mildly ReducedAntibodies

The anti-CEACAM5 humanized MAb, hMN-14, the anti-CD22 humanized MAb,hLL2, the anti-CD20 humanized MAb, hA20, the anti-EGP-1 humanized MAb,hRS7, and anti-mucin humanized MAb, hPAM4, were used in these studies.Each antibody was reduced with dithiothreitol (DTT), used in a50-to-70-fold molar excess, in 40 mM PBS, pH 7.4, containing 5.4 mMEDTA, in a 37° C. bath for 45 mM. The reduced product was purified bysize-exclusion chromatography and/or diafiltration, and wasbuffer-exchanged into a suitable buffer at pH 6.5. The thiol content wasdetermined by Ellman's assay, and was in the 6.5-to-8.5 SH/IgG range.Alternatively, the antibodies were reduced with Tris (2-carboxyethyl)phosphine (TCEP) in phosphate buffer at a pH in the range of 5 to 7,followed by in situ conjugation. The reduced MAb was reacted with˜10-to-15-fold molar excess of CL6-SN-38 of Example 1, or CL7-SN-38 ofExample 2, or CL6-SN-38-10-O—CO₂Et of Example 3, or CL7-SN-38-10—O-CO₂Etof Example 4, or CL2A-SN-38 of Example 10, or CL2E-SN-38 of Example 11using DMSO at 7-15% v/v as a co-solvent, and incubating for 20 min atambient temperature. The conjugate was purified by centrifuged SEC,passage through a hydrophobic column, and finally byultrafiltration-diafiltration. The product was assayed for SN-38 byabsorbance at 366 nm and correlating with standard values, while theprotein concentration was deduced from absorbance at 280 nm, correctedfor spillover of SN-38 absorbance at this wavelength. The SN-38/MAbsubstitution ratios were determined. The purified conjugates were storedas lyophilized formulations in glass vials, capped under vacuum andstored in a −20° C. freezer. SN-38 molar substitution ratios (MSR)obtained for some of these conjugates, which were typically in the 5 to7 range, are shown in Table 7.

TABLE 7 SN-38/MAb Molar substitution ratios (MSR) in some conjugates MAbConjugate MSR hMN-14 hMN-14-[CL2A-SN-38], using drug-linker of 6.1Example 10 hMN-14-[CL6-SN-38], using drug-linker of Example 1 6.8hMN-14-[CL7-SN-38], using drug-linker of Example 2 5.9hMN-14-[CL7-SN-38-10-O—CO₂Et], using 5.8 drug-linker of Example 4hMN-14-[CL2E-SN-38], using drug-linker of 5.9 Example 11 hRS7hRS7-CL2A-SN-38 using drug-linker of Example 10 5.8 hRS7-CL7-SN-38 usingdrug-linker of Example 2 5.9 hRS7-CL7-SN-38 (Et) using drug-linker ofExample 4 6.1 hA20 hA20-CL2A-SN-38 using drug-linker of Example 10 5.8hLL2 hLL2-CL2A-SN-38 using drug-linker of Example 10 5.7 hPAM4hPAM4-CL2A-SN-38 using drug-linker of Example 10 5.9

Example 13 Lyophilized Preparations of SN-38 Immunoconjugates

In a preferred embodiment of the immunoconjugates described in Example12 above, the purified conjugates are contained in the pH range of 5.5to 7.5 in any of the following Good's biological buffers derived from:2-(N-morpholino)ethanesulfonic acid (MES),N-(2-acetamido)-2-iminodiacetic acid (ADA),1,4-piperazinediethanesulfonic acid (PIPES),N-(2-acetamido)-2-aminoethanesulfonic acid (ACES),N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), andN-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) or HEPES. Themost preferred buffer is 25 mM MES, pH 6.5. In an exemplary preparationof a lyophilized conjugate, a solution (69 mL; 16.78 mg/mL proteinconcentration) of the purified CL2A-SN-38 conjugate of the anti-CD22antibody, hLL2, namely hLL2-CL2A-SN-38, in 25 mM MES, pH 6.5 buffer, wasdiluted with 34.1 mL of the same buffer, and mixed with 11.6 mL of 250mM trehalose and 1.16 mL of 1% polysorbate 80. The formulated solutionwas aliquotted in 50-mg aliquots in various vials, and lyophilized. Thevials containing lyophilized immunoconjugate were sealed under vacuum,and stored at 2-8° C. in a refrigerator. The lyophilized immunoconjugatewas stable under these conditions.

Example 14 In Vivo Therapeutic Efficacies in Preclinical Models of HumanPancreatic or Colon Carcinoma

Immune-compromised athymic nude mice (female), bearing subcutaneoushuman pancreatic or colon tumor xenografts were treated with eitherspecific CL2A-SN-38 conjugate or control conjugate or were leftuntreated. The therapeutic efficacies of the specific conjugates wereobserved. FIG. 1 shows a Capan 1 pancreatic tumor model, whereinspecific CL2A-SN-38 conjugates of hRS7 (anti-EGP-1), hPAM4 (anti-mucin),and hMN-14 (anti-CEACAM5) antibodies showed better efficacies thancontrol hA20-CL2A-SN-38 conjugate (anti-CD20) and untreated control.Similarly in a BXPC3 model of human pancreatic cancer, the specifichRS7-CL2A-SN-38 showed better therapeutic efficacy than controltreatments (FIG. 2). Likewise, in an aggressive LS174T model of humancolon carcinoma, treatment with specific hMN-14-CL2A-SN-38 was moreefficacious than control treatment (FIG. 3).

Example 15 Humanized Anti-CD22 MAb (Epratuzumab) Conjugated with SN-38,Alone or in Combination with Anti-CD20 MAb (Veltuzumab), Shows PotentEfficacy for Therapy of Hematologic Tumors

Monoclonal antibody therapy has had a significant impact on themanagement of B-cell malignancies, but is most often used in combinationwith chemotherapy. We developed an antibody-drug conjugate (ADC) thatcombines SN-38, the active component of irinotecan, a topoisomerase Iinhibitor, with an internalizing, humanized anti-CD22 IgG (epratuzumab)and determined its activity alone and in combination with an anti-CD20antibody (veltuzumab).

The CD22 antigen is expressed in most NHL and ALL specimens. Epratuzumab(humanized anti-CD22 MAb) is an internalizing antibody that has beenshown to be safe and therapeutically effective as a naked antibody aloneand in combination with rituximab. Epratuzumab is currently beingstudied in pediatric ALL, as well as a radioconjugate in NHL. (See,e.g., Leonard et al., 2008, Cancer 113:2714-23; Raetz et al., 2008, JClin Oncol 26:3756-62; Morschhauser et al., 2010, J Clin Oncol28:3709-16). Therapeutic use of epratuzumab is not limited to B cellmalignancies, but has also been proposed for autoimmune diseases such assystemic lupus erythematosus (SLE) (Domer et al., 2006, Arthritis ResTher 8:R74; Daridon et a1., 2010, Arthritis Res Ther 12:R204).

Veltuzumab (humanized anti-CD20) has shown anti-proliferative, apoptoticand ADCC effects in vitro similar to rituximab, but with significantlyslower off-rates and increased CDC in several human lymphoma cell lines(see, e.g., U.S. Pat. No. 7,919,273, incorporated herein by referencefrom Col. 34, line 15 to Col. 72, line 2). Very low doses of veltuzumab,given either intravenously or subcutaneously, depleted B cells in normalcynomolgus monkeys and controlled tumor growth in mice bearing humanlymphomas. Veltuzumab has been clinically studied in over 150 patientswith lymphomas and autoimmune diseases. In non-Hodgkin lymphoma (NHL),infusions of 80-750 mg/m² were well tolerated when given once-weekly forfour doses, with the only toxicity being transient mild-moderateinfusion reactions. Objective tumor responses, including durablecomplete responses, occurred at all dose levels. Subcutaneous injectionsof low doses (80-320 mg) have also proved to be safe andpharmacologically active, producing objective responses, includingdurable complete responses, at rates comparable to those reported withrituximab, in patients with NHL and immune thrombocytopenia. (See, e.g.,Goldenberg et al., 2010, J Clin Oncol 28:3709-16; Goldenberg et al.,2010, Leuk Lymphoma 51:747-55; Negrea et al., 2011, Haematologica96:567-73; Goldenberg et al., 2009, Blood 113:1062-70; Morchhauser etal., 2009, J Clin Oncol 27:3346-53; Sharkey et al., 2009, J Nucl Med50:444-53.)

Epratuzumab was conjugated with SN-38 (E-SN-38) at a mole ratio of ˜6:1,using a CL2A linker as described in Examples 10 and 12 above.Conjugation had no effect on the affinity of the antibody for the targetantigen (data not shown). The conjugate was designed to be releasedslowly in the presence of serum (50% released over ˜1.5 days), allowingliberation of the drug when internalized, but also being releasedlocally after binding to the tumor. In vitro and in vivo studies wereperformed to assess the activity of the conjugate against severalsubcutaneously- or intravenously-inoculated B-cell lymphoma cell lines.In vivo studies also examined combination therapy using E-SN-38 and theanti-CD20 antibody veltuzumab (V).

In vitro studies in 4 B-cell lymphoma cells lines (Daudi, Raji, Ramos,WSU-FSCCL) and 4 acute lymphoblastic lymphoma cell lines (697, REH,MN-60 and RS4; 11) expressing varying amounts of CD22 showed an IC₅₀ forE-SN-38 in the nanomolar range, confirming potent activity. As shown inTable 8, the IC₅₀ for E-SN-38 did not strictly correlate with CD22expression or with saensitivity to non-targeted SN-38.

TABLE 8 Expression of CD20 and CD22 and in vitro cytotoxicity of SN-38and E-SN-38 CD20 Expression CD22 Expression Cell % % SN-38 E-SN-38 LineFACS positive FACS positive IC₅₀ (nM) IC₅₀ (nM) Raji 283.6 99.3 47.789.3 1.42 2.10 Ramos 27.2 91.6 7.1 2.2 0.40 2.92 Daudi 18.7 20.3 10.73.1 0.13 0.52 WSU- 8.8 7.8 6.2 0.8 0.50 0.68 FSCCL REH 92.0 55.1 52.999.7 0.47 1.22 697 12.0 22.0 42.2 100 2.23 2.67 RS4; 11 8.4 15.0 34.394.5 2.28 1.68 MN-60 N.D. N.D. 1.23 3.65

Nude or SCID mice were implanted SC with Ramos cells (Burkitt'slymphoma) or IV with WSU-FSCCL (follicular lymphoma, FIG. 4, FIG. 5) or697 (ALL, FIG. 6) cell lines. All doses of immunoconjugates or drugswere given intraperitoneally, twice weekly for 4 weeks. Irinotecan(CPT-11) was administered at the same mole equivalent as theantibody-conjugated SN-38. All dose levels were well tolerated in mice,with toxicity only found at doses of 2×30 mg (2×1500 mg/kg) For Ramos,the endpoint was time to progression to 3.0 cm³ tumor size. ForWSU-FSCCL and 697, therapy was started 5 days after tumor cellinjection. The end of study was progression to hind-leg paralysis, 20%or greater loss in body weight, or other signs of stress. Statisticalanalysis was by log-rank test.

E-SN-38 was active in mice at a dose of 2×0.5 mg weekly for 4 weeks (50mg/kg per week). Toxicology studies in monkeys and rabbits of otherIgG-SN-38 conjugatesw have found a human equivalent of 40 mg/kg/week tobe non-toxic, which is approximately 25 mg SN-38 equivalents/m². Thus,the therapeutic window of emab-SN-38 is at least 10:1.

Nude mice bearing SC Ramos human lymphoma had significant selectiveanti-tumor activity compared to a control, non-targeting, IgG-SN-38conjugate, at a dosing regimen of 75 to 250 μg of the conjugates giventwice-weekly for 4 weeks (FIG. 7). Responses improved in adose-dependent manner for both the specific and irrelevantimmunoconjugates.

Significant anti-tumor activity was also found in several other celllines. When combined with veltuzumab, significant improvement intherapeutic activity was observed (FIG. 4, FIG. 5). For example, mediansurvival in a WSU-FSCCL human follicular B-cell lymphoma IV model withtreatment initiated 5 days after implantation was 41d (0/10 surviving at160d) and 91d (2/10 surviving at 160 d) for untreated andveltuzumab-treated animals, respectively; 63 d (0/10 surviving after 160d) and >160 d (6/10 surviving after 160 d) for E-SN-38 and E-SN-38+V,respectively; and 63 d (0/10) and 91d (2/10) for non-targeting IgG-SN-38conjugate alone and combined with V, respectively. The E-SN-38 conjugatecombined with V was significantly better than all other treatment orcontrol groups (P 0.05). We conclude that E-SN-38 ADC is a potenttherapeutic, even at non-toxic dose levels, and shows significantlyenhanced efficacy when combined with anti-CD20 immunotherapy,representing an important new ADC treatment regimen for B cell diseases.

Example 16 Preparation of hLL1, hLL2, hA20 and hL243 Antibodies

The hLL1, hLL2, hA20 and hL243 antibodies were prepared as previouslydescribed and as summarized below. The constant region sequences of eachof the hLL1, hLL2, hA20 and hL243 antibodies are as shown below in SEQID NO:14 (heavy chain constant region amino acid sequence); SEQ IDNO:134 (heavy chain constant region DNA sequence); SEQ ID NO:135 (lightchain constant region amino acid sequence); and SEQ ID NO:136 (lightchain constant region amino acid sequence). Although the constant regionsequences are derived from the hLL2 antibody, they are identical in eachof hLL1, hLL2, hA20 and hL243. Therefore, each of the hLL1, hLL2, hA20and hL243 antibodies is a G1m3 allotype antibody.

Heavy chain constant region amino acid sequence (CH1-Hinge-CH2—CH3)(SEQ ID NO: 14)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHeavy chain constant region DNA sequence (SEQ ID NO: 134)GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCG GGTAAALight chain constant region amino acid sequence (SEQ ID NO: 135)TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLight chain constant region DNA sequence (SEQ ID NO: 136)ACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

hLL1 Antibody

The hLL1 anti-CD74 antibody was prepared as described in U.S. Pat. No.7,772,373 (incorporated by reference from Col. 3, line 54 to Col. 5,line 32 and Col. 34, line 15 to Col. 40, line 45, FIGS. 1A, 1B, 2A, 2B,3A, 3B, 4A, 4B). The variable region sequences of the light and heavychains of the hLL1 antibody are as described in U.S. Pat. No. 7,772,373(e.g., FIG. 3 and FIG. 4).

A modified strategy as described by Leung et al. (1994, Hybridoma13:469-76) was used to construct the VK and VH genes for hLL1 using acombination of long oligonucleotide synthesis and PCR. For theconstruction of the hLL1 VH domain, two long oligonucleotides, hLL1VHA(176 mer) and hLL1VHB (165-mer) (U.S. Pat. No. 7,772,373) weresynthesized on an automated DNA synthesizer. The hLL1VHA sequencerepresented nt 20 to 195 of the hLL1VH domain. The hLL1 VHB sequencerepresented the minus strand of the hLL1 VH domain complementary to nt173 to 337. The 3′-terminal sequences (22 nt residues) of hLL1VHA and Bwere complementary to each other. Under PCR condition, the 3′-ends ofhLL1 VHA and B annealed to form a short double stranded DNA. Eachannealed end served as a primer for the transcription of single strandedDNA, resulting in a double strand DNA composed of the nt 20 to 337 ofhLL1VH. This DNA was further amplified in the presence of two shortoligonucleotides, hLL1VHBACK and hLL1VHFOR (U.S. Pat. No. 7,772,373) toform the full-length hLL1VH. Double-stranded PCR-amplified product forhLL1VH was gel-purified, restriction-digested with PstI and BstEII andcloned into the complementary PstI/BstEII sites of the heavy chainstaging vector, VHpBS2.

For constructing the full length DNA of the humanized VK sequence,hLL1VKA (159-mer) and hLL1VKB (169-mer) (U.S. Pat. No. 7,772,373) weresynthesized. The hLL1 VKA sequence represented nt 16 to 174 of thehLL1VK domain. The hLL1VKB sequence represented the minus strand of thehLL1VK domain complementary to nt 153 to 321. hLL1VKA and B wereamplified by two short oligonucleotides hLL1VKBACK and hLL1VKFOR (U.S.Pat. No. 7,772,373) to form double-stranded DNA. Further amplificationproduced the full length VK gene (U.S. Pat. No. 7,772,373). Gel-purifiedPCR products for hLL1 VK were restriction-digested with PvuII and BglI11and cloned into the complementary PvuJJBclI sites of the light chainstaging vector, VKpBR2.

The final expression vector hLL1pdHL2 was constructed by sequentiallysubcloning the XbaI-BamHI and XhoI/BamHI fragments of hLL1VK and VH,respectively, into pdHL2. The pdHL2 vector is known in the art (see,e.g., Gillies et al., 1989, J Immunol Methods 125:191). The pdHL2 vectorprovides expression of both IgG heavy and light chain genes that areindependently controlled by two metallothionine promoters and IgHenhancers. Use of pdHL2 as an expression vector for antibody productionhas been disclosed, for example, in Losman et al., 1999, Clin Cancer Res5:3101s-05s.

The fragment containing the VK sequence of hLL1, together with thesignal peptide sequence, was excised from LL1VKpBR2 by doublerestriction digestion with XbaI and BamHI. The ˜550 by VK fragment wasthen subcloned into the XbaI/BamHI site of a mammalian expressionvector, pdHL2. The resulting vector was designated as hLL1VKpdHL2.Similarly, the ˜750 by fragment encoding hLL1 VH, together with thesignal peptide sequence, was excised from LL1VHpBS2 by XhoI and BamHIdigestion and isolated by electrophoresis in an agarose gel. Thefragment was subcloned into the XhoI and HindIII site of hLL1VKpdHL2with the aid of linker comparable to both BamHI and HindIII ends,resulting in the final expression vector, designated as hLL1pdHL2.

Approximately 30 μg of hLL1pdHL2 was linearized by digestion with Sal Iand transfected into Sp2/0-Ag14 cells by electroporation. Thetransfected cells were plated into 96-well plate for 2 days and thenselected for MTX resistance. Supernatants from colonies survivingselection were monitored for chimeric antibody secretion by ELISA assay.Positive cell clones were expanded and hLL1 was purified from cellculture supernatant.

hLL2 Antibody

The hLL2 anti-CD22 antibody was prepared as described in U.S. Pat. No.6,187,287 (incorporated by reference from Col. 3, line 35 to Col. 4,line 34 and Col. 11, line 40 to Col. 20, line 38, FIGS. 1, 4A, 4B, 5A,5B). The variable region sequences of the light and heavy chains of thehLL2 antibody are as described in U.S. Pat. No. 6,187,287 (e.g., FIG. 1,FIG. 5). The LL2 antibody was deposited on May 27, 2005, with theAmerican Type Culture Collection, Manassas, Va. (ATCC Accession No.PTA-6735), formerly the EPB-2 monoclonal antibody, which was producedagainst human Raji cells derived from a Burkitt lymphoma.(Pawlak-Byczkowska et al., 1989, Cancer Res. 49:4568.) The cloning,transfection and protein production were performed as described abovefor the hLL1 antibody.

hA20 Antibody

The hA20 anti-CD20 antibody was prepared as described in U.S. Pat. No.7,919,273 (incorporated by reference from Col. 7, line 25 to Col. 9,line 4 and Col. 34, line 15 to Col. 72, line 2, FIGS. 1A, 1B, 2A, 2B,3A, 3B). The variable region sequences of the light and heavy chains ofthe hA20 antibody are as described in U.S. Pat. No. 7,919,273 (e.g.,FIG. 2, FIG. 3). The cloning, transfection and protein production wereperformed as described above for the hLL1 antibody.

hL243 Antibody

The hL243 anti-HLA-DR antibody was prepared as described in U.S. Pat.No. 7,612,180 (incorporated by reference from Col. 4, line 16 to Col. 6,line 38 and Col. 46, line 50 to Col. 60, line 67, FIGS. 1 to 6). Thevariable region sequences of the light and heavy chains of the hL243antibody are as described in U.S. Pat. No. 7,612,180 (e.g., FIG. 3, FIG.4, FIG. 5, FIG. 6). The cloning, transfection and protein productionwere performed as described above for the hLL1 antibody.

Other known antibodies, such as hPAM4 (U.S. Pat. No. 7,282,567), hA19(U.S. Pat. No. 7,109,304), hIMMU31 (U.S. Pat. No. 7,300,655), hMu-9(U.S. Pat. No. 7,387,773), hMN-14 (U.S. Pat. No. 6,676,924), hMN-15(U.S. Pat. No. 7,541,440), hR1 (U.S. patent application Ser. No.12/689,336), hRS7 (U.S. Pat. No. 7,238,785), hMN-3 (U.S. Pat. No.7,541,440), 15B8 (anti-CD40, U.S. Pat. No. 7,820,170), AB-PG1-XG1-026(U.S. patent application Ser. No. 11/983,372, deposited as ATCC PTA-4405and PTA-4406) and D2/B (WO 2009/130575), may be prepared as describedabove, using the techniques disclosed herein.

Example 17 Preparation of Dock-and-Lock (DNL) Constructs

DDD and AD Fusion Proteins

The DNL technique can be used to make dimers, trimers, tetramers,hexamers, etc. comprising virtually any antibody, antibody fragment,immunomodulator, cytokine, enzyme, peptide, PEG moiety, toxin,xenoantigen or other effector moiety. For certain preferred embodiments,antibodies, cytokines or toxins (such as ranpirnase) may be produced asfusion proteins comprising either a dimerization and docking domain(DDD) or anchoring domain (AD) sequence. Although in preferredembodiments the DDD and AD moieties may be joined to antibodies,antibody fragments, cytokines, toxins or other effector moieties asfusion proteins, the skilled artisan will realize that other methods ofconjugation exist, such as chemical cross-linking, click chemistryreaction, etc.

The technique is not limiting and any protein or peptide of use may beproduced as an AD or DDD fusion protein for incorporation into a DNLconstruct. Where chemical cross-linking is utilized, the AD and DDDconjugates may comprise any molecule that may be cross-linked to an ADor DDD sequence using any cross-linking technique known in the art. Incertain exemplary embodiments, a dendrimer or other polymeric moietysuch as polyethyleneimine or polyethylene glycol (PEG), may beincorporated into a DNL construct, as described in further detail below.

Expression Vectors

The plasmid vector pdHL2 has been used to produce a number of antibodiesand antibody-based constructs. See Gillies et al., J Immunol Methods(1989), 125:191-202; Losman et al., Cancer (Phila) (1997), 80:2660-6.The di-cistronic mammalian expression vector directs the synthesis ofthe heavy and light chains of IgG. The vector sequences are mostlyidentical for many different IgG-pdHL2 constructs, with the onlydifferences existing in the variable domain (V_(H) and V_(L)) sequences.Using molecular biology tools known to those skilled in the art, theseIgG expression vectors can be converted into Fab-DDD or Fab-ADexpression vectors.

To generate Fab-DDD expression vectors, the coding sequences for thehinge, CH2 and CH3 domains of the heavy chain were replaced with asequence encoding the first 4 residues of the hinge, a 14 residueGly-Ser linker and a DDD moiety, such as the first 44 residues of humanRIIα (referred to as DDD1, SEQ ID NO:15). To generate Fab-AD expressionvectors, the sequences for the hinge, CH2 and CH3 domains of IgG werereplaced with a sequence encoding the first 4 residues of the hinge, a15 residue Gly-Ser linker and an AD moiety, such as a 17 residuesynthetic AD called AKAP-IS (referred to as AD1, SEQ ID NO:17), whichwas generated using bioinformatics and peptide array technology andshown to bind RIIα dimers with a very high affinity (0.4 nM). See Alto,et al. Proc. Natl. Acad. Sci., U.S.A (2003), 100:4445-50.

Two shuttle vectors were designed to facilitate the conversion ofIgG-pdHL2 vectors to either Fab-DDD1 or Fab-AD1 expression vectors, asdescribed below.

Preparation of CH1

The CH1 domain was amplified by PCR using the pdHL2 plasmid vector as atemplate. The left PCR primer consisted of the upstream (5′) end of theCH1 domain and a SacII restriction endonuclease site, which is 5′ of theCH1 coding sequence. The right primer consisted of the sequence codingfor the first 4 residues of the hinge followed by four glycines and aserine, with the final two codons (GS) comprising a Barn HI restrictionsite. The 410 by PCR amplimer was cloned into the PGEMT® PCR cloningvector (PROMEGA®, Inc.) and clones were screened for inserts in the T7(5′) orientation.

A duplex oligonucleotide was synthesized to code for the amino acidsequence of DDD 1 preceded by 11 residues of the linker peptide, withthe first two codons comprising a BamHI restriction site. A stop codonand an EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below.

(SEQ ID NO: 137) GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

Two oligonucleotides, designated RIIA1-44 top and RIIA1-44 bottom, whichoverlap by 30 base pairs on their 3′ ends, were synthesized and combinedto comprise the central 154 base pairs of the 174 by DDD1 sequence. Theoligonucleotides were annealed and subjected to a primer extensionreaction with Taq polymerase. Following primer extension, the duplex wasamplified by PCR. The amplimer was cloned into PGEMT® and screened forinserts in the T7 (5′) orientation.

A duplex oligonucleotide was synthesized to code for the amino acidsequence of AD1 preceded by 11 residues of the linker peptide with thefirst two codons comprising a BamHI restriction site. A stop codon andan EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below.

(SEQ ID NO: 138) GSGGGGSGGGGSQIEYLAKQIVDNAIQQA

Two complimentary overlapping oligonucleotides encoding the abovepeptide sequence, designated AKAP-IS Top and AKAP-IS Bottom, weresynthesized and annealed. The duplex was amplified by PCR. The amplimerwas cloned into the PGEMT® vector and screened for inserts in the T7(5′) orientation.

Ligating DDD1 with CH1

A 190 by fragment encoding the DDD1 sequence was excised from PGEMT®with BamHI and NotI restriction enzymes and then ligated into the samesites in CH1-PGEMT® to generate the shuttle vector CH1-DDD1-PGEMT®.

Ligating AD1 with CH1

A 110 by fragment containing the AD1 sequence was excised from PGEMT®with BamHI and NotI and then ligated into the same sites in CH1-PGEMT®to generate the shuttle vector CH1-AD1-PGEMTa

Cloning CH1-DDD1 or CH1-AD1 into pdHL2-Based Vectors

With this modular design either CH1-DDD1 or CH1-AD1 can be incorporatedinto any IgG construct in the pdHL2 vector. The entire heavy chainconstant domain is replaced with one of the above constructs by removingthe SacII/EagI restriction fragment (CH1-CH3) from pdHL2 and replacingit with the SacII/EagI fragment of CH1-DDD1 or CH1-AD1, which is excisedfrom the respective PGEMT® shuttle vector.

Construction of h679-Fd-AD1-pdHL2

h679-Fd-AD1-pdHL2 is an expression vector for production of h679 Fabwith AD1 coupled to the carboxyl terminal end of the CH1 domain of theFd via a flexible Gly/Ser peptide spacer composed of 14 amino acidresidues. A pdHL2-based vector containing the variable domains of h679was converted to h679-Fd-AD1-pdHL2 by replacement of the SacII/EagIfragment with the CH1-AD1 fragment, which was excised from theCH1-AD1-SV3 shuttle vector with SacII and EagI.

Production and Purification of h679-Fab-AD1

The h679-Fd-AD1-pdHL2 vector was linearized by digestion with Sal Irestriction endonuclease and transfected into Sp/EEE myeloma cells (U.S.Pat. No. 7,785,880) by electroporation. The di-cistronic expressionvector directs the synthesis and secretion of both h679 kappa lightchain and h679 Fd-AD 1, which combine to form h679 Fab-AD 1. Followingelectroporation, the cells were plated in 96-well tissue culture platesand transfectant clones were selected with 0.05 μM methotrexate (MTX).Clones were screened for protein expression by ELISA using microtiterplates coated with a BSA-IMP260 (HSG) conjugate and detection withHRP-conjugated goat anti-human Fab. BIACORE® analysis using an HSG(IMP239) sensorchip was used to determine the productivity by measuringthe initial slope obtained from injection of diluted media samples. Thehighest producing clone had an initial productivity of approximately 30mg/L. A total of 230 mg of h679-Fab-AD1 was purified from 4.5 liters ofroller bottle culture by single-step IMP291 affinity chromatography.Culture media was concentrated approximately 10-fold by ultrafiltrationbefore loading onto an IMP291-affigel column. The column was washed tobaseline with PBS and h679-Fab-AD1 was eluted with 1 M imidazole, 1 mMEDTA, 0.1 M NaAc, pH 4.5. SE-HPLC analysis of the eluate shows a singlesharp peak with a retention time consistent with a 50 kDa protein (notshown). Only two bands, which represent the polypeptide constituents ofh679-AD1, were evident by reducing SDS-PAGE analysis (not shown).

Construction of C-DDD1-Fd-hMN-14-pdHL2

C-DDD1-Fd-hMN-14-pdHL2 is an expression vector for production of astable dimer that comprises two copies of a fusion proteinC-DDD1-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at the carboxylterminus of CH1 via a flexible peptide spacer. The plasmid vectorhMN-14(I)-pdHL2, which has been used to produce hMN-14 IgG, wasconverted to C-DDD1-Fd-hMN-14-pdHL2 by digestion with SacII and EagIrestriction endonucleases to remove the CH1-CH3 domains and insertion ofthe CH1-DDD1 fragment, which was excised from the CH1-DDD1-SV3 shuttlevector with SacII and EagI.

The same technique has been utilized to produce plasmids for Fabexpression of a wide variety of known antibodies, such as hLL1, hLL2,hPAM4, hR1, hRS7, hMN-14, hMN-15, hA19, hA20 and many others. Generally,the antibody variable region coding sequences were present in a pdHL2expression vector and the expression vector was converted for productionof an AD- or DDD-fusion protein as described above. The AD- andDDD-fusion proteins comprising a Fab fragment of any of such antibodiesmay be combined, in an approximate ratio of two DDD-fusion proteins perone AD-fusion protein, to generate a trimeric DNL construct comprisingtwo Fab fragments of a first antibody and one Fab fragment of a secondantibody.

Production and Purification of C-DDD1-Fab-hMN-14

The C-DDD1-Fd-hMN-14-pdHL2 vector was transfected into Sp2/0-derivedmyeloma cells by electroporation. C-DDD1-Fd-hMN-14-pdHL2 is adi-cistronic expression vector, which directs the synthesis andsecretion of both hMN-14 kappa light chain and hMN-14 Fd-DDD1, whichcombine to form C-DDD1-hMN-14 Fab. The fusion protein forms a stablehomodimer via the interaction of the DDD1 domain.

Following electroporation, the cells were plated in 96-well tissueculture plates and transfectant clones were selected with 0.05 μMmethotrexate (MTX). Clones were screened for protein expression by ELISAusing microtiter plates coated with WI2 (a rat anti-id monoclonalantibody to hMN-14) and detection with HRP-conjugated goat anti-humanFab. The initial productivity of the highest producing C-DDD1-Fab-hMN14Fab clone was 60 mg/L.

Affinity Purification of C-DDD1-hMN-14 with AD1-Affigel

The DDD/AD interaction was utilized to affinity purify DDD1-containingconstructs. AD1-C is a peptide that was made synthetically consisting ofthe AD1 sequence and a carboxyl terminal cysteine residue, which wasused to couple the peptide to Affigel following reaction of thesulfhydryl group with chloroacetic anhydride. DDD-containing dimerstructures specifically bind to the AD1-C-Affigel resin at neutral pHand can be eluted at low pH (e.g., pH 2.5).

A total of 81 mg of C-DDD1-Fab-hMN-14 was purified from 1.2 liters ofroller bottle culture by single-step AD1-C affinity chromatography.Culture media was concentrated approximately 10-fold by ultrafiltrationbefore loading onto an AD1-C-affigel column. The column was washed tobaseline with PBS and C-DDD1-Fab-hMN-14 was eluted with 0.1 M Glycine,pH 2.5. SE-HPLC analysis of the eluate showed a single protein peak witha retention time consistent with a 107 kDa protein (not shown). Thepurity was also confirmed by reducing SDS-PAGE, showing only two bandsof molecular size expected for the two polypeptide constituents ofC-DDD1-Fab-hMN-14 (not shown).

The binding activity of C-DDD1-Fab-hMN-14 was determined by SE-HPLCanalysis of samples in which the test article was mixed with variousamounts of WI2. A sample prepared by mixing WI2 Fab andC-DDD1-Fab-hMN-14 at a molar ratio of 0.75:1 showed three peaks, whichwere attributed to unbound C-DDD1-Fab-hMN14 (8.71 min),C-DDD1-Fab-hMN-14 bound to one WI2 Fab (7.95 min), and C-DDD1-Fab-hMN14bound to two WI2 Fabs (7.37 min) (not shown). When a sample containingWI2 Fab and C-DDD1-Fab-hMN-14 at a molar ratio of 4 was analyzed, only asingle peak at 7.36 minutes was observed (not shown). These resultsdemonstrated that hMN14-Fab-DDD1 is dimeric and has two active bindingsites. Very similar results were obtained when this experiment wasrepeated with an hMN-14 Fab construct with DDD1 linked to the aminoterminal instead of the carboxyl terminal end (not shown).

A competitive ELISA demonstrated that C-DDD1-Fab-hMN-14 binds to CEAwith an avidity similar to hMN-14 IgG, and significantly stronger thanmonovalent hMN-14 Fab (not shown). ELISA plates were coated with afusion protein containing the epitope (A3B3) of CEA for which hMN-14 isspecific.

C-DDD2-Fd-hMN-14-pdHL2

C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production ofC-DDD2-Fab-hMN-14, which possesses a dimerization and docking domainsequence of DDD2 (SEQ ID NO:16) appended to the carboxyl terminus of theFd of hMN-14 via a 14 amino acid residue Gly/Ser peptide linker. Thefusion protein secreted is composed of two identical copies of hMN-14Fab held together by non-covalent interaction of the DDD2 domains.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides, which comprise the coding sequence forpart of the linker peptide and residues 1-13 of DDD2, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and PstI, respectively.

The duplex DNA was ligated with the shuttle vector CH1-DDD1-PGEMT®,which was prepared by digestion with BamHI and PstI, to generate theshuttle vector CH1-DDD2-PGEMT®. A 507 by fragment was excised fromCH1-DDD2-PGEMT® with SacII and EagI and ligated with the IgG expressionvector hMN-14(I)-pdHL2, which was prepared by digestion with SacII andEagI. The final expression construct was designatedC-DDD2-Fd-hMN-14-pdHL2. Similar techniques have been utilized togenerated DDD2-fusion proteins of the Fab fragments of a number ofdifferent humanized antibodies.

h679-Fd-AD2-pdHL2

h679-Fab-AD2, was designed to pair to C-DDD2-Fab-hMN-14.h679-Fd-AD2-pdHL2 is an expression vector for the production ofh679-Fab-AD2, which possesses an anchoring domain sequence of AD2 (SEQID NO:18) appended to the carboxyl terminal end of the CH1 domain via a14 amino acid residue Gly/Ser peptide linker. AD2 has one cysteineresidue preceding and another one following the anchor domain sequenceof AD 1.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides (AD2 Top and AD2 Bottom), which comprisethe coding sequence for AD2 and part of the linker sequence, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and SpeI, respectively.

The duplex DNA was ligated into the shuttle vector CH1-AD1-PGEMT®, whichwas prepared by digestion with BamHI and SpeI, to generate the shuttlevector CH1-AD2-PGEMT®. A 429 base pair fragment containing CH1 and AD2coding sequences was excised from the shuttle vector with SacII and EagIrestriction enzymes and ligated into h679-pdHL2 vector that prepared bydigestion with those same enzymes. The final expression vector ish679-Fd-AD2-pdHL2.

Example 18 Generation of Trimeric DNL Constructs

TF2 DNL Construct

A trimeric DNL construct designated TF2 was obtained by reactingC-DDD2-Fab-hMN-14 with h679-Fab-AD2. A pilot batch of TF2 was generatedwith >90% yield as follows. Protein L-purified C-DDD2-Fab-hMN-14 (200mg) was mixed with h679-Fab-AD2 (60 mg) at a 1.4:1 molar ratio. Thetotal protein concentration was 1.5 mg/ml in PBS containing 1 mM EDTA.Subsequent steps involved TCEP reduction, HIC chromatography, DMSOoxidation, and IMP 291 affinity chromatography. Before the addition ofTCEP, SE-HPLC did not show any evidence of a₂b formation. Addition of 5mM TCEP rapidly resulted in the formation of a₂b complex consistent witha 157 kDa protein expected for the binary structure. TF2 was purified tonear homogeneity by IMP 291 affinity chromatography (not shown). IMP 291is a synthetic peptide containing the HSG hapten to which the 679 Fabbinds (Rossi et al., 2005, Clin Cancer Res 11:7122s-29s). SE-HPLCanalysis of the IMP 291 unbound fraction demonstrated the removal of a₄,a₂ and free kappa chains from the product (not shown).

The functionality of TF2 was determined by BIACORE® assay. TF2,C-DDD1-hMN-14+h679-AD1 (used as a control sample of noncovalent a₂bcomplex), or C-DDD2-hMN-14+h679-AD2 (used as a control sample ofunreduced a₂ and b components) were diluted to 1 μg/ml (total protein)and passed over a sensorchip immobilized with HSG. The response for TF2was approximately two-fold that of the two control samples, indicatingthat only the h679-Fab-AD component in the control samples would bind toand remain on the sensorchip. Subsequent injections of WI2 IgG, ananti-idiotype antibody for hMN-14, demonstrated that only TF2 had aDDD-Fab-hMN-14 component that was tightly associated with h679-Fab-AD asindicated by an additional signal response. The additional increase ofresponse units resulting from the binding of WI2 to TF2 immobilized onthe sensorchip corresponded to two fully functional binding sites, eachcontributed by one subunit of C-DDD2-Fab-hMN-14. This was confirmed bythe ability of TF2 to bind two Fab fragments of WI2 (not shown).

TF10 DNL Construct

A similar protocol was used to generate a trimeric TF10 DNL construct,comprising two copies of a C-DDD2-Fab-hPAM4 and one copy ofC-AD2-Fab-679. The TF10 bispecific ([hPAM4]₂×h679) antibody was producedusing the method disclosed for production of the (anti CEA)₂×anti HSGbsAb TF2, as described above. The TF10 construct bears two humanizedPAM4 Fabs and one humanized 679 Fab.

The two fusion proteins (hPAM4-DDD2 and h679-AD2) were expressedindependently in stably transfected myeloma cells. The tissue culturesupernatant fluids were combined, resulting in a two-fold molar excessof hPAM4-DDD2. The reaction mixture was incubated at room temperaturefor 24 hours under mild reducing conditions using 1 mM reducedglutathione. Following reduction, the DNL reaction was completed by mildoxidation using 2 mM oxidized glutathione. TF10 was isolated by affinitychromatography using IMP291-affigel resin, which binds with highspecificity to the h679 Fab.

Example 19 Production of AD- and DDD-Linked Fab and IgG Fusion Proteinsfrom Multiple Antibodies

Using the techniques described in the preceding Examples, the IgG andFab fusion proteins shown in Table 7 were constructed and incorporatedinto DNL constructs. The fusion proteins retained the antigen-bindingcharacteristics of the parent antibodies and the DNL constructsexhibited the antigen-binding activities of the incorporated antibodiesor antibody fragments.

TABLE 7 Fusion proteins comprising IgG or Fab Fusion Protein BindingSpecificity C-AD1-Fab-h679 HSG C-AD2-Fab-h679 HSG C-(AD)₂-Fab-h679 HSGC-AD2-Fab-h734 Indium-DTPA C-AD2-Fab-hA20 CD20 C-AD2-Fab-hA20L CD20C-AD2-Fab-hL243 HLA-DR C-AD2-Fab-hLL2 CD22 N-AD2-Fab-hLL2 CD22C-AD2-IgG-hMN-14 CEACAM5 C-AD2-IgG-hR1 IGF-1R C-AD2-IgG-hRS7 EGP-1C-AD2-IgG-hPAM4 MUC C-AD2-IgG-hLL1 CD74 C-DDD1-Fab-hMN-14 CEACAM5C-DDD2-Fab-hMN-14 CEACAM5 C-DDD2-Fab-h679 HSG C-DDD2-Fab-hA19 CD19C-DDD2-Fab-hA20 CD20 C-DDD2-Fab-hAFP AFP C-DDD2-Fab-hL243 HLA-DRC-DDD2-Fab-hLL1 CD74 C-DDD2-Fab-hLL2 CD22 C-DDD2-Fab-hMN-3 CEACAM6C-DDD2-Fab-hMN-15 CEACAM6 C-DDD2-Fab-hPAM4 MUC C-DDD2-Fab-hR1 IGF-1RC-DDD2-Fab-hRS7 EGP-1 N-DDD2-Fab-hMN-14 CEACAM5

Example 20 Antibody-Dendrimer DNL Complex for siRNA

Cationic polymers, such as polylysine, polyethylenimine, orpolyamidoamine (PAMAM)-based dendrimers, form complexes with nucleicacids. However, their potential applications as non-viral vectors fordelivering therapeutic genes or siRNAs remain a challenge. One approachto improve selectivity and potency of a dendrimeric nanoparticle may beachieved by conjugation with an antibody that internalizes upon bindingto target cells.

We synthesized and characterized a novel immunoconjugate, designatedE1-G5/2, which was made by the DNL method to comprise half of ageneration 5 (G5) PAMAM dendrimer (G5/2) site-specifically linked to astabilized dimer of Fab derived from hRS7, a humanized antibody that israpidly internalized upon binding to the Trop-2 antigen expressed onvarious solid cancers.

Methods

E1-G5/2 was prepared by combining two self-assembling modules, AD2-G5/2and hRS7-Fab-DDD2, under mild redox conditions, followed by purificationon a Protein L column. To make AD2-G5/2, we derivatized the AD2 peptidewith a maleimide group to react with the single thiol generated fromreducing a G5 PAMAM with a cystamine core and used reversed-phase HPLCto isolate AD2-G5/2. We produced hRS7-Fab-DDD2 as a fusion protein inmyeloma cells, as described in the Examples above.

The molecular size, purity and composition of E1-G5/2 were analyzed bysize-exclusion HPLC, SDS-PAGE, and Western blotting. The biologicalfunctions of E1-G5/2 were assessed by binding to an anti-idiotypeantibody against hRS7, a gel retardation assay, and a DNase protectionassay.

Results

E1-G5/2 was shown by size-exclusion HPLC to consist of a major peak(>90%) flanked by several minor peaks (not shown). The threeconstituents of E1-G5/2 (Fd-DDD2, the light chain, and AD2-G5/2) weredetected by reducing SDS-PAGE and confirmed by Western blotting (notshown). Anti-idiotype binding analysis revealed E1-G5/2 contained apopulation of antibody-dendrimer conjugates of different size, all ofwhich were capable of recognizing the anti-idiotype antibody, thussuggesting structural variability in the size of the purchased G5dendrimer (not shown). Gel retardation assays showed E1-G5/2 was able tomaximally condense plasmid DNA at a charge ratio of 6:1 (+/−), with theresulting dendriplexes completely protecting the complexed DNA fromdegradation by DNase I (not shown).

CONCLUSION

The DNL technique can be used to build dendrimer-based nanoparticlesthat are targetable with antibodies. Such agents have improvedproperties as carriers of drugs, plasmids or siRNAs for applications invitro and in vivo. In preferred embodiments, anti-B-cell antibodies,such as anti-CD22 and/or anti-CD20, may be utilized to deliver cytotoxicor cytostatic siRNA species to targeted B-cells for therapy of lymphoma,leukemia, autoimmune or other diseases and conditions.

Example 21 Targeted Delivery of siRNA Using Protamine Linked Antibodies

Summary

RNA interference (RNAi) has been shown to down-regulate the expressionof various proteins such as HER2, VEGF, Raf-1, bcl-2, EGFR and numerousothers in preclinical studies. Despite the potential of RNAi to silencespecific genes, the full therapeutic potential of RNAi remains to berealized due to the lack of an effective delivery system to target cellsin vivo.

To address this critical need, we developed novel DNL constructs havingmultiple copies of human protamine tethered to a tumor-targeting,internalizing hRS7 (anti-Trop-2) antibody for targeted delivery ofsiRNAs in vivo. A DDD2-L-thP1 module comprising truncated humanprotamine (thP1, residues 8 to 29 of human protamine 1) was produced, inwhich the sequences of DDD2 and thP1 were fused respectively to the N-and C-terminal ends of a humanized antibody light chain (not shown). Thesequence of the truncated hP1 (thP1) is shown below. Reaction ofDDD2-L-thP1 with the antibody hRS7-IgG-AD2 under mild redox conditions,as described in the Examples above, resulted in the formation of anE1-L-thP1 complex (not shown), comprising four copies of thP1 attachedto the carboxyl termini of the hRS7 heavy chains.

tHP1 (SEQ ID NO: 139) RSQSRSRYYRQRQRSRRRRRRS

The purity and molecular integrity of E1-L-thP1 following Protein Apurification were determined by size-exclusion HPLC and SDS-PAGE (notshown). In addition, the ability of E1-L-thP1 to bind plasmid DNA orsiRNA was demonstrated by the gel shift assay (not shown). E1-L-thP1 waseffective at binding short double-stranded oligonucleotides (not shown)and in protecting bound DNA from digestion by nucleases added to thesample or present in serum (not shown).

The ability of the E1-L-thP1 construct to internalize siRNAs intoTrop-2-expressing cancer cells was confirmed by fluorescence microscopyusing FITC-conjugated siRNA and the human Calu-3 lung cancer cell line(not shown).

Methods

The DNL technique was employed to generate E1-L-thP1. The hRS7 IgG-ADmodule, constructed as described in the Examples above, was expressed inmyeloma cells and purified from the culture supernatant using Protein Aaffinity chromatography. The DDD2-L-thP1 module was expressed as afusion protein in myeloma cells and was purified by Protein L affinitychromatography. Since the CH3-AD2-IgG module possesses two AD2 peptidesand each can bind to a DDD2 dimer, with each DDD2 monomer attached to aprotamine moiety, the resulting E1-L-thP1 conjugate comprises fourprotamine groups. E1-L-thp1 was formed in nearly quantitative yield fromthe constituent modules and was purified to near homogeneity (not shown)with Protein A.

DDD2-L-thP1 was purified using Protein L affinity chromatography andassessed by size exclusion HPLC analysis and SDS-PAGE under reducing andnonreducing conditions (data not shown). A major peak was observed at9.6 mM (not shown). SDS-PAGE showed a major band between 30 and 40 kDain reducing gel and a major band about 60 kDa (indicating a dimeric formof DDD2-L-thP1) in nonreducing gel (not shown). The results of Westernblotting confirmed the presence of monomeric DDD2-L-tP1 and dimericDDD2-L-tP1 on probing with anti-DDD antibodies (not shown).

To prepare the E1-L-thP1, hRS7-IgG-AD2 and DDD2-L-thP1 were combined inapproximately equal amounts and reduced glutathione (final concentration1 mM) was added. Following an overnight incubation at room temperature,oxidized glutathione was added (final concentration 2 mM) and theincubation continued for another 24 h. E1-L-thP1 was purified from thereaction mixture by Protein A column chromatography and eluted with 0.1M sodium citrate buffer (pH 3.5). The product peak (not shown) wasneutralized, concentrated, dialyzed with PBS, filtered, and stored inPBS containing 5% glycerol at 2 to 8° C. The composition of E1-L-thP1was confirmed by reducing SDS-PAGE (not shown), which showed thepresence of all three constituents (AD2-appended heavy chain,DDD2-L-htP1, and light chain).

The ability of DDD2-L-thP1 and E1-L-thP1 to bind DNA was evaluated bygel shift assay. DDD2-L-thP1 retarded the mobility of 500 ng of a linearform of 3-kb DNA fragment in 1% agarose at a molar ratio of 6 or higher(not shown). E1-L-thP1 retarded the mobility of 250 ng of a linear200-bp DNA duplex in 2% agarose at a molar ratio of 4 or higher (notshown), whereas no such effect was observed for hRS7-IgG-AD2 alone (notshown). The ability of E1-L-thP1 to protect bound DNA from degradationby exogenous DNase and serum nucleases was also demonstrated (notshown).

The ability of E1-L-thP1 to promote internalization of bound siRNA wasexamined in the Trop-2 expressing ME-180 cervical cell line (not shown).Internalization of the E1-L-thP1 complex was monitored using FITCconjugated goat anti-human antibodies. The cells alone showed nofluorescence (not shown). Addition of FITC-labeled siRNA alone resultedin minimal internalization of the siRNA (not shown). Internalization ofE1-L-thP1 alone was observed in 60 minutes at 37° C. (not shown).E1-L-thP1 was able to effectively promote internalization of boundFITC-conjugated siRNA (not shown). E1-L-thP1 (10 μg) was mixed withFITC-siRNA (300 nM) and allowed to form E1-L-thP1-siRNA complexes whichwere then added to Trop-2-expressing Calu-3 cells. After incubation for4 h at 37° C. the cells were checked for internalization of siRNA byfluorescence microscopy (not shown).

The ability of E1-L-thP1 to induce apoptosis by internalization of siRNAwas examined. E1-L-thP1 (10 μg) was mixed with varying amounts of siRNA(AllStars Cell Death siRNA, Qiagen, Valencia, Calif.). TheE1-L-thP1-siRNA complex was added to ME-180 cells. After 72 h ofincubation, cells were trypsinized and annexin V staining was performedto evaluate apoptosis. The Cell Death siRNA alone or E1-L-thP1 alone hadno effect on apoptosis (not shown). Addition of increasing amounts ofE1-L-thP1-siRNA produced a dose-dependent increase in apoptosis (notshown). These results show that E1-L-thP1 could effectively deliversiRNA molecules into the cells and induce apoptosis of target cells.

Conclusions

The DNL technology provides a modular approach to efficiently tethermultiple protamine molecules to the anti-Trop-2 hRS7 antibody resultingin the novel molecule E1-L-thPl. SDS-PAGE demonstrated the homogeneityand purity of E1-L-thPl. DNase protection and gel shift assays showedthe DNA binding activity of E1-L-thPl. E1-L-thP1 internalized in thecells like the parental hRS7 antibody and was able to effectivelyinternalize siRNA molecules into Trop-2-expressing cells, such as ME-180and Calu-3.

The skilled artisan will realize that the DNL technique is not limitedto any specific antibody or siRNA species. Rather, the same methods andcompositions demonstrated herein can be used to make targeted deliverycomplexes comprising any antibody, any siRNA carrier and any siRNAspecies. The use of a bivalent IgG in targeted delivery complexes wouldresult in prolonged circulating half-life and higher binding avidity totarget cells, resulting in increased uptake and improved efficacy.

Example 22 Ribonuclease Based DNL Immunotoxins Comprising QuadrupleRanpirnase (Rap) Conjugated to B-Cell Targeting Antibodies

We applied the DNL method to generate a novel class of immunotoxins,each of which comprises four copies of Rap site-specifically linked to abivalent IgG. We combined a recombinant Rap-DDD module, produced in E.coli, with recombinant, humanized IgG-AD modules, which were produced inmyeloma cells and targeted B-cell lymphomas and leukemias via binding toCD20 (hA20, veltuzumab), CD22 (hLL2, epratuzumab) or HLA-DR (hL243,IMMU-114), to generate 20-Rap, 22-Rap and C2-Rap, respectively. For eachconstruct, a dimer of Rap was covalently tethered to the C-terminus ofeach heavy chain of the respective IgG. A control construct, 14-Rap, wasmade similarly, using labetuzumab (hMN-14), that binds to an antigen(CEACAM5) not expressed on B-cell lymphomas/leukemias.

Rap-DDD2 (SEQ ID NO: 140)pQDWLTFQKKHITNTRDVDCDNIMSTNLFHCKDKNTFIYSRPEPVKAICKGIIASKNVLTTSEFYLSDCNVTSRPCKYKLKKSTNKFCVTCENQAPVHFVGVGSC GGGGSLE CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA VEHHHHHH

The deduced amino acid sequence of secreted Rap-DDD2 is shown above (SEQID NO:140). Rap, underlined; linker, italics; DDD2, bold; pQ,amino-terminal glutamine converted to pyroglutamate. Rap-DDD2 wasproduced in E. coli as inclusion bodies, which were purified by IMACunder denaturing conditions, refolded and then dialyzed into PBS beforepurification by anion exchange chromatography. SDS-PAGE under reducingconditions resolved a protein band with a Mr appropriate for Rap-DDD2(18.6 kDa) (not shown). The final yield of purified Rap-DDD2 was 10 mg/Lof culture.

The DNL method was employed to rapidly generate a panel of IgG-Rapconjugates. The IgG-AD modules were expressed in myeloma cells andpurified from the culture supernatant using Protein A affinitychromatography. The Rap-DDD2 module was produced and mixed with IgG-AD2to form a DNL complex. Since the CH3-AD2-IgG modules possess two AD2peptides and each can tether a Rap dimer, the resulting IgG-Rap DNLconstruct comprises four Rap groups and one IgG. IgG-Rap is formednearly quantitatively from the constituent modules and purified to nearhomogeneity with Protein A.

Prior to the DNL reaction, the CH3-AD2-IgG exists as both a monomer, anda disulfide-linked dimer (not shown). Under non-reducing conditions, theIgG-Rap resolves as a cluster of high molecular weight bands of theexpected size between those for monomeric and dimeric CH3-AD2-IgG (notshown). Reducing conditions, which reduces the conjugates to theirconstituent polypeptides, show the purity of the IgG-Rap and theconsistency of the DNL method, as only bands representingheavy-chain-AD2 (HC-AD2), kappa light chain and Rap-DDD2 were visualized(not shown). Reversed phase HPLC analysis of 22-Rap (not shown) resolveda single protein peak at 9.10 min eluting between the two peaks ofCH3-AD2-IgG-hLL2, representing the monomeric (7.55 min) and the dimeric(8.00 min) forms. The Rap-DDD2 module was isolated as a mixture of dimerand tetramer (reduced to dimer during DNL), which were eluted at 9.30and 9.55 min, respectively (not shown).

LC/MS analysis of 22-Rap (not shown) showed that both the Rap-DDD2 andHC-AD2 polypeptides have an amino terminal glutamine that is convertedto pyroglutamate (pQ) and that 22-Rap has 6 of its 8 constituentpolypeptides modified by pQ.

In vitro cytotoxicity was evaluated in three NHL cell lines. Each cellline expresses CD20 at a considerably higher surface density compared toCD22; however, the internalization rate for hLL2 (anti-CD22) is muchfaster than hA20 (anti-CD20). 14-Rap shares the same structure as 22-Rapand 20-Rap, but its antigen (CEACAM5) is not expressed by the NHL cells.Cells were treated continuously with IgG-Rap as single agents or withcombinations of the parental MAbs plus rRap. Both 20-Rap and 22-Rapkilled each cell line at concentrations above 1 nM, indicating thattheir action is cytotoxic as opposed to merely cytostatic (not shown).20-Rap was the most potent IgG-Rap, suggesting that antigen density maybe more important than internalization rate. Similar results wereobtained for Daudi and Ramos, where 20-Rap (EC50˜0.1 nM) was 3-6-foldmore potent than 22-Rap (not shown). The rituximab-resistant mantle celllymphoma line, Jeko-1, exhibits increased CD20 but decreased CD22,compared to Daudi and Ramos. Importantly, 20-Rap exhibited very potentcytotoxicity (EC₅₀˜20 pM) in Jeko-1, which was 25-fold more potent than22-Rap (not shown).

The DNL method provides a modular approach to efficiently tethermultiple cytotoxins onto a targeting antibody, resulting in novelimmunotoxins that are expected to show higher in vivo potency due toimproved pharmacokinetics and targeting specificity. Targeting Rap witha MAb to a cell surface antigen enhanced its tumor-specificcytotoxicity. Antigen density and internalization rate are both criticalfactors for the observed in vitro potency of IgG-Rap. In vitro resultsshow that CD20-, CD22-, or HLA-DR-targeted IgG-Rap have potent biologicactivity for therapy of B-cell lymphomas and leukemias. The skilledartisan will realize that the modular DNL technique may be utilized toproduce Rap DNL constructs targeted to CD22.

Example 23 Production and Use of a DNL Construct Comprising TwoDifferent Antibody Moieties and a Cytokine

In certain embodiments, trimeric DNL constructs may comprise threedifferent effector moieties, for example two different antibody moietiesand a cytokine moiety. We report here the generation andcharacterization of the first bispecific MAb-IFNα, designated 20-C2-2b,which comprises two copies of IFN-α2b and a stabilized F(ab)₂ of hL243(humanized anti-HLA-DR; IMMU-114) site-specifically linked to veltuzumab(humanized anti-CD20). In vitro, 20-C2-2b inhibited each of fourlymphoma and eight myeloma cell lines, and was more effective thanmonospecific CD20-targeted MAb-IFNα or a mixture comprising the parentalantibodies and IFNα in all but one (HLA-DR⁻/CD20⁻) myeloma line (notshown), suggesting that 20-C2-2b should be useful in the treatment ofvarious hematopoietic disorders. The 20-C2-2b displayed greatercytotoxicity against KMS12-BM (CD20⁺/HLA-DR⁺ myeloma) than monospecificMAb-IFNa that targets only HLA-DR or CD20 (not shown), indicating thatall three components in 20-C2-2b can contribute to toxicity. Ourfindings indicate that a given cell's responsiveness to MAb-IFNα dependson its sensitivity to IFNα and the specific antibodies, as well as theexpression and density of the targeted antigens.

Because 20-C2-2b has antibody-dependent cellular cytotoxicity (ADCC),but not CDC, and can target both CD20 and HLA-DR, it is useful fortherapy of a broad range of hematopoietic disorders that express eitheror both antigens.

Antibodies

The abbreviations used in the following discussion are: 20(C_(H)3-AD2-IgG-v-mab, anti-CD20 IgG DNL module); C2(C_(H)1-DDD2-Fab-hL243, anti-HLA-DR Fab₂ DNL module); 2b (dimericIFNα2B-DDD2 DNL module); 734 (anti-in-DTPA IgG DNL module used asnon-targeting control). The following MAbs were provided byImmunomedics, Inc.: veltuzumab or v-mab (anti-CD20 IgG₁), hL243γ4p(Immu-114, anti-HLA-DR IgG₄), a murine anti-IFNα MAb, and ratanti-idiotype MAbs to v-mab (WR2) and hL243 (WT).

DNL Constructs

Monospecific MAb-IFNa (20-2b-2b,734-2b-2b and C2-2b-2b) and thebispecific HexAb (20-C2-C2) were generated by combination of anIgG-AD2-module with DDD2-modules using the DNL method, as described inthe preceding Examples. The 734-2b-2b, which comprises tetrameric IFNα2band MAb h734 [anti-Indium-DTPA IgG₁], was used as a non-targetingcontrol MAb-IFNα.

The construction of the mammalian expression vector as well as thesubsequent generation of the production clones and the purification ofC_(H)3-AD2-IgG-v-mab are disclosed in the preceding Examples. Theexpressed recombinant fusion protein has the AD2 peptide linked to thecarboxyl terminus of the C_(H)3 domain of v-mab via a 15 amino acid longflexible linker peptide. Co-expression of the heavy chain-AD2 and lightchain polypeptides results in the formation of an IgG structure equippedwith two AD2 peptides. The expression vector was transfected into Sp/ESFcells (an engineered cell line of Sp2/0) by electroporation. The pdHL2vector contains the gene for dihydrofolate reductase, thus allowingclonal selection, as well as gene amplification with methotrexate (MTX).Stable clones were isolated from 96-well plates selected with mediacontaining 0.2 μM MTX. Clones were screened for C_(H)3-AD2-IgG-vmabproductivity via a sandwich ELISA. The module was produced in rollerbottle culture with serum-free media.

The DDD-module, IFNα2b-DDD2, was generated as discussed above byrecombinant fusion of the DDD2 peptide to the carboxyl terminus of humanIFNα2b via an 18 amino acid long flexible linker peptide. As is the casefor all DDD-modules, the expressed fusion protein spontaneously forms astable homodimer.

The C_(H)1-DDD2-Fab-hL243 expression vector was generated fromhL243-IgG-pdHL2 vector by excising the sequence for theC_(H)1-H1nge-C_(H)2-C_(H)3 domains with SacII and EagI restrictionenzymes and replacing it with a 507 by sequence encoding C_(H)1-DDD2,which was excised from the C-DDD2-hMN-14-pdHL2 expression vector withthe same enzymes. Following transfection of C_(H)1-DDD2-Fab-hL243-pdHL2into Sp/ESF cells by electroporation, stable, MTX-resistant clones werescreened for productivity via a sandwich ELISA using 96-well microtiterplates coated with mouse anti-human kappa chain to capture the fusionprotein, which was detected with horseradish peroxidase-conjugated goatanti-human Fab. The module was produced in roller bottle culture.

Roller bottle cultures in serum-free H-SFM media and fed-batchbioreactor production resulted in yields comparable to other IgG-AD2modules and cytokine-DDD2 modules generated to date.C_(H)3-AD2-IgG-v-mab and IFNα2b-DDD2 were purified from the culturebroths by affinity chromatography using MABSELECT™ (GE Healthcare) andHIS-SELECT® HF Nickel Affinity Gel (Sigma), respectively, as describedpreviously (Rossi et al., Blood 2009, 114:3864-71). The culture brothcontaining the C_(H)1-DDD2-Fab-hL243 module was applied directly toKAPPASELECT® affinity gel (GE-Healthcare), which was washed to baselinewith PBS and eluted with 0.1 M Glycine, pH 2.5.

The purity of the DNL modules was assessed by SDS-PAGE and SE-HPLC (notshown). Analysis under non-reducing conditions showed that, prior to theDNL reaction, IFNα2b-DDD2 and C_(H)1-DDD2-Fab-hL243 exist asdisulfide-linked dimers (not shown). This phenomenon, which is alwaysseen with DDD-modules, is beneficial, as it protects the reactivesulfhydryl groups from irreversible oxidation. In comparison,C_(H)3-AD2-IgG-v-mab (not shown) exists as both a monomer and adisulfide-linked dimer, and is reduced to monomer during the DNLreaction. Reducing SDS-PAGE demonstrated that each module was purifiedto near homogeneity and identified the component polypeptides comprisingeach module (not shown).

Generation of 20-C2-2b by DNL

Three DNL modules (C_(H)3-AD2-IgG-v-mab, C_(H)1-DDD2-Fab-hL243, andIFN-α2b-DDD2) were combined in equimolar quantities to generate thebsMAb-IFNa, 20-C2-2b. Following an overnight docking step under mildreducing conditions (1 mM reduced glutathione) at room temperature,oxidized glutathione was added (2 mM) to facilitate disulfide bondformation (locking). The 20-C2-2b was purified to near homogeneity usingthree sequential affinity chromatography steps, first with Protein A(MABSELECT™), second by IMAC using HIS-SELECT® HF Nickel Affinity Gel,and third by an hL243-anti-idiotype affinity chromatography. Only thoseDNL constructs comprising each of the 3 desired monomers bound to allthree columns.

The skilled artisan will realize that affinity chromatography may beused to purify DNL complexes comprising any combination of effectormoieties, so long as ligands for each of the three effector moieties canbe obtained and attached to the column material. The selected DNLconstruct is the one that binds to each of three columns containing theligand for each of the three effector moieties and can be eluted afterwashing to remove unbound complexes.

Generation and Characterization of 20-C2-2b

The bispecific MAb-IFNα was generated by combining the IgG-AD2 module,C_(H)3-AD2-IgG-v-mab, with two different dimeric DDD-modules,C_(H)1-DDD2-Fab-hL243 and IFNα2b-DDD2. Due to the random association ofeither DDD-module with the two AD2 groups, two side-products, 20-C2-C2and 20-2b-2b are expected to form, in addition to 20-C2-2b.

Non-reducing SDS-PAGE (not shown) resolved 20-C2-2b (−305 kDa) as acluster of bands positioned between those of 20-C2-C2 (−365 kDa) and20-2b-2b (255 kDa). Reducing SDS-PAGE resolved the five polypeptides(v-mab HC-AD2, hL243 Fd-DDD2, IFNα2b-DDD2 and co-migrating v-mab andhL243 kappa light chains) comprising 20-C2-2b (not shown). IFNα2b-DDD2and hL243 Fd-DDD2 are absent in 20-C2-C2 and 20-2b-2b. MABSELECT™ bindsto all three of the major species produced in the DNL reaction, butremoves any excess IFNα2b-DDD2 and C_(H)1-DDD2-Fab-hL243. TheHIS-SELECT® unbound fraction contained mostly 20-C2-C2 (not shown). Theunbound fraction from WT affinity chromatography comprised 20-2b-2b (notshown). Each of the samples was subjected to SE-HPLC andimmunoreactivity analyses, which corroborated the results andconclusions of the SDS-PAGE analysis.

SE-HPLC analysis of 20-C2-2b resolved a predominant protein peak with aretention time (6.7 min) consistent with its calculated mass and betweenthose of the larger 20-C2-C2 (6.6 min) and smaller 20-2b-2b (6.85 min),as well as some higher molecular weight peaks that likely representnon-covalent dimers formed via self-association of IFNα2b (not shown).

Immunoreactivity assays demonstrated the homogeneity of 20-C2-2b witheach molecule containing the three functional groups (not shown).Incubation of 20-C2-2b with an excess of antibodies to any of the threeconstituent modules resulted in quantitative formation of high molecularweight immune complexes and the disappearance of the 20-C2-2b peak (notshown). The MAb-IFNa showed similar binding avidity to their parentalMAbs (not shown).

IFNα Biological Activity

The specific activities for various MAb-IFNα were measured using acell-based reporter gene assay and compared to peginterferon alfa-2b(not shown). Expectedly, the specific activity of 20-C2-2b (2454IU/pmol), which has two IFNα2b groups, was significantly lower thanthose of 20-2b-2b (4447 IU/pmol) or 734-2b-2b (3764 IU/pmol), yetgreater than peginterferon alfa-2b (P<0.001) (not shown). The differencebetween 20-2b-2b and 734-2b-2b was not significant. The specificactivity among all agents varies minimally when normalized to IU/pmol oftotal IFNa. Based on these data, the specific activity of each IFNα2bgroup of the MAb-IFNa is approximately 30% of recombinant IFNn2.b (4000IU/pmol).

In the ex-vivo setting, the 20-C2-2b DNL construct depleted lymphomacells more effectively than normal B cells and had no effect on T cells(not shown). However, it did efficiently eliminate monocytes (notshown). Where v-mab had no effect on monocytes, depletion was observedfollowing treatment with hL243α4p and MAb-IFNα, with 20-2b-2b and734-2b-2b exhibiting similar toxicity (not shown). Therefore, thepredictably higher potency of 20-C2-2b is attributed to the combinedactions of anti-HLA-DR and IFNα, which may be augmented by HLA-DRtargeting.

The skilled artisan will realize that the approach described here toproduce and use bispecific immunocytokine, or other DNL constructscomprising three different effector moieties, may be utilized with anycombinations of antibodies, antibody fragments, cytokines or othereffectors that may be incorporated into a DNL construct, for example thecombination of anti-CD20 and anti-CD22 with IFNα2b.

Example 24 Hexavalent DNL Constructs

The DNL technology described above for formation of trivalent DNLcomplexes was applied to generate hexavalent IgG-based DNL structures(HIDS). Because of the increased number of binding sites for targetantigens, hexavalent constructs are expected to show greater affinityand/or efficacy against target cells. Two types of modules, which wereproduced as recombinant fusion proteins, were combined to generate avariety of HIDS. Fab-DDD2 modules were as described above. The Fab-DDD2modules form stable homodimers that bind to AD2-containing modules. Togenerate HIDS, C-H-AD2-IgG modules were created to pair with theFab-DDD2 modules.

C-H-AD2-IgG modules have an AD2 peptide fused to the carboxyl terminus(C) of the heavy (H) chain of IgG via a peptide linker. The DNA codingsequences for the linker peptide followed by the AD2 peptide are coupledto the 3′ end of the CH3 (heavy chain constant domain 3) coding sequenceby standard recombinant DNA methodologies, resulting in a contiguousopen reading frame. When the heavy chain-AD2 polypeptide is co-expressedwith a light chain polypeptide, an IgG molecule is formed possessing twoAD2 peptides, which can therefore bind two Fab-DDD2 dimers. TheC-H-AD2-IgG module can be combined with any Fab-DDD2 module to generatea wide variety of hexavalent structures composed of an Fc fragment andsix Fab fragments. If the C-H-AD2-IgG module and the Fab-DDD2 module arederived from the same parental monoclonal antibody (MAb) the resultingHIDS is monospecific with 6 binding arms to the same antigen. If themodules are instead derived from two different MAbs then the resultingHIDS are bispecific, with two binding arms for the specificity of theC-H-AD2-IgG module and 4 binding arms for the specificity of theFab-DDD2 module.

The same technique has been utilized to produce DNL complexes comprisingan IgG moiety attached to four effector moieties, such as cytokines. Inan exemplary embodiment, an IgG moiety was attached to four copies ofinterferon-α2b. The antibody-cytokine DNL construct exhibited superiorpharmacokinetic properties and/or efficacy compared to PEGylated formsof interferon-α2b.

Creation of C-H-AD2-IgG-pdHL2 Expression Vectors

The pdHL2 mammalian expression vector has been used to mediate theexpression of many recombinant IgGs. A plasmid shuttle vector wasproduced to facilitate the conversion of any IgG-pdHL2 vector into aC-H-AD2-IgG-pdHL2 vector. The gene for the Fc (CH2 and CH3 domains) wasamplified using the pdHL2 vector as a template and a pair of primers.The amplimer was cloned in the PGEMT® PCR cloning vector. The Fc insertfragment was excised from PGEMT® with XbaI and BamHI restriction enzymesand ligated with AD2-pdHL2 vector that was prepared by digestion ofh679-Fab-AD2-pdHL2 with XbaI and BamHI, to generate the shuttle vectorFc-AD2-pdHL2.

To convert any IgG-pdHL2 expression vector to a C-H-AD2-IgG-pdHL2expression vector, an 861 by BsrGI/NdeI restriction fragment is excisedfrom the former and replaced with a 952 by BsrGI/NdeI restrictionfragment excised from the Fc-AD2-pdHL2 vector. BsrGI cuts in the CH3domain and NdeI cuts downstream (3′) of the expression cassette.

Production of C-H-AD2-hLL2 IgG

Epratuzumab, or hLL2 IgG, is a humanized anti-human CD22 MAb. Anexpression vector for C-H-AD2-hLL2 IgG was generated from hLL2IgG-pdHL2, as described above, and used to transfect Sp2/0 myeloma cellsby electroporation. Following transfection, the cells were plated in96-well plates and transgenic clones were selected in media containingmethotrexate. Clones were screened for C-H-AD2-hLL2 IgG productivity bya sandwich ELISA using 96-well microtiter plates coated with anhLL2-specific anti-idiotype MAb and detection with peroxidase-conjugatedanti-human IgG. Clones were expanded to roller bottles for proteinproduction and C-H-AD2-hLL2 IgG was purified from the spent culturemedia in a single step using Protein-A affinity chromatography. SDS-PAGEanalysis demonstrated that the purified C-H-AD2-hLL2-IgG consisted ofboth monomeric and disulfide-linked dimeric forms of the module (notshown). Protein bands representing these two forms are evident bySDS-PAGE under non-reducing conditions, while under reducing conditionsall of the forms are reduced to two bands representing the constituentpolypeptides (Heavy chain-AD2 and kappa chain) (not shown). No othercontaminating bands were detected.

Production of C-H-AD2-hA20 IgG

hA20 IgG is a humanized anti-human CD20 MAb. An expression vector forC-H-AD2-hA20 IgG was generated from hA20 IgG-pDHL2, as described above,and used to transfect Sp2/0 myeloma cells by electroporation. Followingtransfection, the cells were plated in 96-well plates and transgenicclones were selected in media containing methotrexate. Clones werescreened for C-H-AD2-hA20 IgG productivity by a sandwich ELISA using96-well microtiter plates coated with a hA20-specific anti-idiotype MAband detection with peroxidase-conjugated anti-human IgG. Clones wereexpanded to roller bottles for protein production and C-H-AD2-hA20 IgGwas purified from the spent culture media in a single step usingProtein-A affinity chromatography. SE-HPLC and SDS-PAGE analyses gavevery similar results to those obtained for C-H-AD2-hLL2 IgG (not shown).

Example 25 Generation of Hexavalent DNL Constructs

Generation of Hex-hA20

The DNL method was used to create Hex-hA20, a monospecific anti-CD20HIDS, by combining C-H-AD2-hA20 IgG with hA20-Fab-DDD2. The Hex-hA20structure contains six anti-CD20 Fab fragments and an Fc fragment,arranged as four Fab fragments and one IgG antibody. Hex-hA20 was madeas described below.

A 210% molar equivalent of (hA20-Fab-DDD2)₂ was mixed with C-H-AD2-hA20IgG. This molar ratio was used because two Fab-DDD2 dimers are coupledto each C-H-AD2-hA20 IgG molecule and an additional 10% excess of theformer to ensure that the coupling reaction is complete. The mixture wastypically made in phosphate buffered saline, pH 7.4 (PBS) with 1 mMEDTA. Then reduced glutathione (GSH) was added to a final concentrationof 1 mM and the solution was held at room temperature (16-25° C.) for1-24 hours. Following reduction, oxidized glutathione (GSSH) was addeddirectly to the reaction mixture to a final concentration of 2 mM andthe solution was held at room temperature for 1-24 hours.

After oxidation, the reaction mixture was loaded directly onto aProtein-A affinity chromatography column. The column was washed with PBSand the Hex-hA20 was eluted with 0.1 M glycine, pH 2.5. Since excesshA20-Fab-DDD2 was used in the reaction, there was no unconjugatedC-H-AD2-hA20 IgG, or incomplete DNL structures containing only one(hA20-Fab-DDD2)₂ moiety. The unconjugated excess hA20-Fab-DDD2 does notbind to the affinity resin. The calculated molecular weight from thededuced amino acid sequences of the constituent polypeptides is 386 kDa.Size exclusion HPLC analysis showed a single protein peak with aretention time consistent with a protein structure of 375-400 kDa (notshown).

Generation of Hex-hLL2

The DNL method was used to create a monospecific anti-CD22 HIDS(Hex-hLL2) by combining C-H-AD2-hLL2 IgG with hLL2-Fab-DDD2. The DNLreaction was accomplished as described above for Hex-hA20. Thecalculated molecular weight from the deduced amino acid sequences of theconstituent polypeptides is 386 kDa. Size exclusion HPLC analysis showeda single protein peak with a retention time consistent with a proteinstructure of 375-400 kDa (not shown). SDS-PAGE analysis undernon-reducing conditions showed a cluster of high molecular weight bands,which were eliminated under reducing conditions to leave only the threeexpected polypeptide chains: HC-AD2, Fd-DDD2, and the kappa chain (notshown).

Generation of DNL1 and DNL1C

The DNL method was used to create bispecific HIDS by combiningC-H-AD2-hLL2 IgG with either hA20-Fab-DDD2 to obtain DNL1 or hMN-14-DDD2to obtain DNL1C. DNL1 has four binding arms for CD20 and two for CD22.As hMN-14 is a humanized MAb to carcinoembryonic antigen (CEACAM5),DNL1C has four binding arms for CEACAM5 and two for CD22. The DNLreactions were accomplished as described for Hex-hA20 above. HPLC andSDS-PAGE were consistent with the desired products.

Generation of DNL2 and DNL2C

The DNL method was used to create bispecific HIDS by combiningC-H-AD2-hA20 IgG with either hLL2-Fab-DDD2 to obtain DNL2 or hMN-14-DDD2to obtain DNL2C. DNL2 has four binding arms for CD22 and two for CD20.DNL2C has four binding arms for CEACAM5 and two for CD20. The DNLreactions were accomplished as described for Hex-hA20. HPLC and SDS-PAGEwere consistent with the desired products.

Stability in Serum

The stability of DNL1 and DNL2 in human serum was determined using abispecific ELISA assay. The protein structures were incubated at 10μg/ml in fresh pooled human sera at 37° C. and 5% CO₂ for five days. Forday 0 samples, aliquots were frozen in liquid nitrogen immediately afterdilution in serum. ELISA plates were coated with an anti-Id to hA20 IgGand bispecific binding was detected with an anti-Id to hLL2 IgG. BothDNL1 and DNL2 were highly stable in serum and maintained completebispecific binding activity (not shown).

Binding Activity

The HIDS generated as described above retained the binding properties oftheir parental Fab/IgGs. Competitive ELISAs were used to investigate thebinding avidities of the various HIDS using either a rat anti-idiotypeMAb to hA20 (WR2) to assess the binding activity of the hA20 componentsor a rat anti-idiotype MAb to hLL2 (WN) to assess the binding activityof the hLL2 components. To assess hA20 binding, ELISA plates were coatedwith hA20 IgG and the HIDS were allowed to compete with the immobilizedIgG for WR2 binding. To assess hLL2 binding, plates were coated withhLL2 IgG and the HIDS were allowed to compete with the immobilized IgGfor WN binding. The relative amount of anti-Id bound to the immobilizedIgG was detected using peroxidase-conjugated anti-Rat IgG.

Examining the relative CD20 binding avidities, DNL2, which has two CD20binding groups, showed a similar binding avidity to hA20 IgG, which alsohas two CD20-binding arms (not shown). DNL1, which has four CD20-bindinggroups, had a stronger (−4-fold) relative avidity than DNL2 or hA20 IgG(not shown). Hex-hA20, which has six CD20-binding groups, had an evenstronger (−10-fold) relative avidity than hA20 IgG (not shown).

Similar results were observed for CD22 binding. DNL1, which has two CD20binding groups, showed a similar binding avidity to hLL2 IgG, which alsohas two CD22-binding arms (not shown). DNL2, which has four CD22-bindinggroups, had a stronger (>5-fold) relative avidity than DNL1 or hLL2 IgG.Hex-hLL2, which has six CD22-binding groups, had an even stronger(>10-fold) relative avidity than hLL2 IgG (not shown). As both DNL2 andDNL3 contain two hA20 Fabs and four hLL2 Fabs, they showed similarstrength in binding to the same anti-id antibody (not shown).

In Vivo Anti-Tumor Activity

The HIDS were shown to have therapeutic efficacy in vivo using a humanBurkitt Lymphoma model in mice. Low doses (12 μg) of DNL2 and Hex-hA20more than doubled the survival times of tumor bearing mice. Treatmentwith higher doses (60 μg) resulted in long-term survivors.

In Vitro Activity

Some of the HIDS were observed to have potent anti-proliferativeactivity on lymphoma cell lines. DNL1, DNL2 and Hex-hA20 inhibited cellgrowth of Daudi Burkitt Lymphoma cells in vitro (not shown). Treatmentof the cells with 10 nM concentrations was substantially more effectivefor the HIDS compared to rituximab (not shown). Using a cell countingassay, the potency of DNL1 and DNL2 was estimated to be more than100-fold greater than that of rituximab, while the Hex-hA20 was shown tobe even more potent (not shown). This was confirmed with an MTSproliferation assay in which dose-response curves were generated forDaudi cells treated with a range of concentrations of the HIDS (notshown). Compared to rituximab, the bispecific HIDS (DNL1 and DNL2) andHex-hA20 were >100-fold and >10000-fold more potent, respectively.

Dose-response curves for HIDS (DNL1, DNL2, Hex-hA20) versus a parent IgG(hA20 IgG) were compared for three different lymphoma cell lines, usingan MTS proliferation assay. In Daudi lymphoma cells, the bispecificstructures DNL1 and DNL2 showed >100-fold more potent anti-proliferativeactivity and Hex-hA20 showed >10.000-fold more potent activity than theparent hA20 IgG (not shown). Hex-hLL2 and the control structures (DNL1-Cand DNL2-C) had very little anti-proliferative activity in this assay(not shown).

In Raji lymphoma cells, Hex-hA20 displayed potent anti-proliferativeactivity, but DNL2 showed only minimal activity compared with hA20 IgG(not shown). In Ramos lymphoma cells, both DNL2 and Hex-hA20 displayedpotent anti-proliferative activity, compared with hA20 IgG (not shown).These results show that the increased potency of HIDS relative to theparent IgGs is not limited to particular cell lines, but rather is ageneral phenomenon for cells displaying the appropriate targets.

CDC and ADCC Activity of Hexavalent DNL Constructs

In vivo, anti-CD20 monoclonal antibodies such as rituximab and hA20 canutilize complement-dependent cytotoxicity (CDC), antibody-dependentcellular cytotoxicity (ADCC) and signal transduction induced growthinhibition/apoptosis for tumor cell killing. The hexavalent DNLstructures (DNL1, DNL2, Hex-hA20) were tested for CDC activity usingDaudi cells in an in vitro assay. Surprisingly, none of the hexavalentstructures that bind CD20 exhibited CDC activity (not shown). The parenthA20 IgG exhibited potent CDC activity (not shown), while as expectedthe hLL2 antibody against CD22 showed no activity (not shown). The lackof effect of DNL2 and Hex-hA20 was of interest, since they comprisehA20-IgG-Ad2, which showed similar positive CDC activity to hA20 IgG(not shown).

DNL1 was assayed for ADCC activity using freshly isolated peripheralblood mononuclear cells. Both rituximab and hA20 IgG showed potentactivity on Daudi cells, while DNL1 did not exhibit any detectable ADCCactivity (not shown).

These data suggest that the Fc region may become inaccessible foreffector functions (CDC and ADCC) when four additional Fab groups aretethered to its carboxyl termini. Therefore, the hexavalent DNLstructures appear to rely only on signal transduction induced growthinhibition/apoptosis for in vivo anti-tumor activity.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention.

What is claimed is:
 1. A method of treating an autoimmune diseasecomprising administering to an individual with an autoimmune disease, animmunoconjugate consisting of (i) an anti-CD22 antibody or antigenbinding fragment thereof; and (ii) at least one therapeutic agentattached by a linker to the anti-CD22 antibody or fragment thereof,wherein the therapeutic agent is selected from the group consisting ofan anti-B cell antibody, an antigen-binding fragment of an anti-B cellantibody, an immunomodulator, a drug, an anti-angiogenic agent, aproapoptotic agent, a cytokine inhibitor, a chemokine inhibitor, atyrosine kinase inhibitor, a sphingosine inhibitor, a hormone, a hormoneantagonist, an enzyme inhibitor, and an oligonucleotide.
 2. The methodof claim 1, wherein the anti-CD22 antibody or fragment thereof comprisesthe light chain complementarity determining region (CDR) sequences CDR1(KSSQSVLYSANHKYLA, SEQ ID NO:1), CDR2 (WASTRES, SEQ ID NO:12), and CDR3(HQYLSSWTF, SEQ ID NO:3) and the heavy chain CDR sequences CDR1 (SYWLH,SEQ ID NO:4), CDR2 (YINPRNDYTEYNQNFKD, SEQ ID NO:5), and CDR3 (RDITTFY,SEQ ID NO:6).
 3. The method of claim 1, wherein the anti-CD22 antibodyor fragment thereof competes with, blocks binding to, or binds to thesame epitope of CD22 as an LL2 antibody comprising the light chain CDRsequences CDR1 (KSSQSVLYSANHKYLA, SEQ ID NO:1), CDR2 (WASTRES, SEQ IDNO:2), and CDR3 (HQYLSSWTF, SEQ ID NO:3) and the heavy chain CDRsequences CDR1 (SYWLH, SEQ ID NO:4), CDR2 (YINPRNDYTEYNQNFKD, SEQ IDNO:5), and CDR3 (RDITTFY, SEQ ID NO:6).
 4. The method of claim 1,wherein the anti-CD22 antibody or fragment thereof is selected from thegroup consisting of epratuzumab, 1F5, H₁B22, FPC1, LT22, MEM-1, RFB4,bu59, fpc1, mc64-12 and IS7.
 5. The method of claim 1, furthercomprising administering to said individual an anti-B cell antibody orantigen-binding fragment thereof that binds to an antigen selected fromthe group consisting of CD5, CD15, CD19, CD20, CD21, CD22, CD23, CD30,CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD74, CD79a, CD80, CD138,HLA-DR and VEGF.
 6. The method of claim 5, wherein the anti-B cellantibody or fragment thereof binds to CD20.
 7. The method of claim 6,wherein the anti-B cell antibody is selected from the group consistingof GA101, BCX-301, DXL 625, L26, B-Ly1, MEM-97, LT20, 2H7, AT80, B-H20,HI20a, HI47, 13.6E12, 4f11, Scl1, 7d1, rituximab and veltuzumab.
 8. Themethod of claim 6, wherein the anti-B cell antibody is rituximab orveltuzumab.
 9. The method of claim 6, wherein the anti-B cell antibodyor fragment thereof competes with, blocks binding to, or binds to thesame epitope of CD20 as an hA20 antibody comprising the light chaincomplementarity-determining region (CDR) sequences CDR1 (RASSSVSYIH; SEQID NO:7), CDR2 (ATSNLAS; SEQ ID NO:8), and CDR3 (QQWTSNPPT; SEQ ID NO:9)and the heavy chain variable region CDR sequences CDR1 (SYNMH; SEQ IDNO:10), CDR2 (AIYPGNGDTSYNQKFKG; SEQ ID NO:11), and CDR3 (STYYGGDWYFDV;SEQ ID NO:12).
 10. The method of claim 6, wherein the anti-B cellantibody or fragment thereof comprises the light chaincomplementarity-determining region (CDR) sequences CDR1 (RASSSVSYIH; SEQID NO:7), CDR2 (ATSNLAS; SEQ ID NO:8), and CDR3 (QQWTSNPPT; SEQ ID NO:9)and the heavy chain variable region CDR sequences CDR1 (SYNMH; SEQ IDNO:10), CDR2 (AIYPGNGDTSYNQKFKG; SEQ ID NO:11), and CDR3 (STYYGGDWYFDV;SEQ ID NO:12).
 11. The method of claim 1, wherein the immunoconjugate isadministered subcutaneously and the therapeutic agent is selected fromthe group consisting of an anti-B cell antibody, an antigen-bindingfragment of an anti-B cell antibody, an immunomodulator and a cytokine.12. The method of claim 1, wherein the anti-B cell antibody or fragmentof an anti-B cell antibody binds to an antigen selected from the groupconsisting of CD5, CD15, CD19, CD20, CD21, CD22, CD23, CD30, CD33, CD37,CD38, CD40, CD40L, CD46, CD52, CD74, CD79a, CD80, CD138, HLA-DR andVEGF.
 13. The method of claim 1, wherein the immunomodulator is selectedfrom the group consisting of cytokines, lymphokines, monokines, stemcell growth factors, lymphotoxins, hematopoietic factors, colonystimulating factors (CSF), interferons (IFN), parathyroid hormone,thyroxine, insulin, proinsulin, relaxin, prorelaxin, folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH),luteinizing hormone (LH), hepatic growth factor, prostaglandin,fibroblast growth factor, prolactin, placental lactogen, OB protein,transforming growth factor (TGF), TGF-α, TGF-β, insulin-like growthfactor (IGF), erythropoietin, thrombopoietin, tumor necrosis factor(TNF), TNF-α, TNF-β, mullerian-inhibiting substance, mousegonadotropin-associated peptide, inhibin, activin, vascular endothelialgrowth factor, integrin, interleukin (IL), granulocyte-colonystimulating factor (G-CSF), granulocyte macrophage-colony stimulatingfactor (GM-CSF), interferon-α, interferon-β, interferon-γ, S1 factor,IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18 IL-21, IL-23,IL-25, LIF, kit-ligand, FLT-3, angiostatin, thrombospondin andendostatin.
 14. The method of claim 13, wherein the immunomodulator isinterferon-α.
 15. The method of claim 1, wherein the drug is selectedfrom the group consisting of aplidin, azaribine, anastrozole,azacytidine, bleomycin, bortezomib, bryostatin-1, busulfan,calicheamycin, camptothecin, 10-hydroxycamptothecin, carmustine,celebrex, chlorambucil, cisplatin, irinotecan (CPT-11), SN-38,carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine,docetaxel, dactinomycin, daunomycin glucuronide, daunorubicin,dexamethasone, diethylstilbestrol, doxorubicin, doxorubicin glucuronide,epirubicin glucuronide, ethinyl estradiol, estramustine, etoposide,etoposide glucuronide, etoposide phosphate, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, fluorouracil,fluoxymesterone, gemcitabine, hydroxyprogesterone caproate, hydroxyurea,idarubicin, ifosfamide, L-asparaginase, leucovorin, lomustine,mechlorethamine, medroprogesterone acetate, megestrol acetate,melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,mithramycin, mitomycin, mitotane, phenyl butyrate, prednisone,procarbazine, paclitaxel, pentostatin, PSI-341, semustine streptozocin,tamoxifen, taxanes, taxol, testosterone propionate, thalidomide,thioguanine, thiotepa, teniposide, topotecan, uracil mustard, velcade,vinblastine, vinorelbine and vincristine.
 16. The method of claim 15,wherein the drug is SN-38.
 17. The method of claim 1, wherein theanti-angiogenic agent is selected from the group consisting ofangiostatin, endostatin, baculostatin, canstatin, maspin, an anti-VEGFbinding molecule, an anti-placental growth factor binding molecule andan anti-vascular growth factor binding molecule.
 18. The method of claim1, wherein the autoimmune disease is selected from the group consistingof acute immune thrombocytopenia, chronic immune thrombocytopenia,dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupuserythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, pemphigus vulgaris, diabetes mellitus(e.g., juvenile diabetes), Henoch-Schonlein purpura, post-streptococcalnephritis, erythema nodosum, Takayasu's arteritis, Addison's disease,rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerativecolitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa,ankylosing spondylitis, Goodpasture's syndrome, thromboangitisobliterans, Sjogren's syndrome, primary biliary cirrhosis, Hashimoto'sthyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, perniciousanemia, rapidly progressive glomerulonephritis, psoriasis and fibrosingalveolitis.
 19. The method of claim 18, wherein the disease is systemiclupus erythematosus (SLE), Sjogren's syndrome or rheumatoid arthritis.20. The method of claim 1, further comprising killing B cells by amechanism selected from the group consisting of homotypic adhesion, lossof mitochondrial membrane potential, production of reactive oxygenspecies, increased phosphorylation of ERKs and JNK, downregulation ofpAkt and Bcl-xL, and enlargement of lysosomes.
 21. The method of claim1, wherein the anti-CD22 antibody is a G1m3 allotype.
 22. The method ofclaim 1, wherein the anti-CD22 antibody is a chimeric, humanized orhuman antibody.
 23. The method of claim 1, wherein the anti-CD22antibody or fragment thereof comprises human IgG1, IgG2, IgG3, or IgG4constant regions.
 24. The method of claim 16, further comprising storingthe immunoconjugate prior to administration at a pH in the range of 5.5to 7.5 in a solution comprising a buffer selected from the groupconsisting of 2-(N-morpholino)ethanesulfonic acid (MES),N-(2-acetamido)-2-iminodiacetic acid (ADA),1,4-piperazinediethanesulfonic acid (PIPES),N-(2-acetamido)-2-aminoethanesulfonic acid (ACES),N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), andN-(2-hydroxyethyppiperazine-N′-(2-ethanesulfonic acid) or HEPES.
 25. Themethod of claim 24, wherein the buffer is 25 mM MES, pH 6.5.
 26. Themethod of claim 24, wherein the solution further comprises trehalose andpolysorbate
 80. 27. The method of claim 26, further comprisinglyophilizing the solution and storing the lyophilized antibody at 2-8°C.
 28. The method of claim 27, wherein the lyophilized antibody isstable at 2-8° C. for at least 12 months.
 29. A method of treating anautoimmune disease comprising administering to an individual with anautoimmune disease, an immunoconjugate consisting of (i) an anti-CD22antibody or antigen binding fragment thereof and (ii) at least onetherapeutic agent attached to the anti-CD22 antibody or fragmentthereof, wherein the therapeutic agent is selected from the groupconsisting of an immunomodulator, a drug, an anti-angiogenic agent, aproapoptotic agent, a cytokine inhibitor, a chemokine inhibitor, atyrosine kinase inhibitor, a sphingosine inhibitor, a hormone, a hormoneantagonist, an enzyme inhibitor and an oligonucleotide.
 30. The methodof claim 29, wherein the anti-CD22 antibody or fragment thereofcomprises the light chain complementarity determining region (CDR)sequences CDR1 (KSSQSVLYSANHKYLA, SEQ ID NO:1), CDR2 (WASTRES, SEQ IDNO:12), and CDR3 (HQYLSSWTF, SEQ ID NO:3) and the heavy chain CDRsequences CDR1 (SYWLH, SEQ ID NO:4), CDR2 (YINPRNDYTEYNQNFKD, SEQ IDNO:5), and CDR3 (RDITTFY, SEQ ID NO:6).
 31. The method of claim 29,wherein the anti-CD22 antibody or fragment thereof competes with, blocksbinding to, or binds to the same epitope of CD22 as an LL2 antibodycomprising the light chain CDR sequences CDR1 (KSSQSVLYSANHKYLA, SEQ IDNO:1), CDR2 (WASTRES, SEQ ID NO:2), and CDR3 (HQYLSSWTF, SEQ ID NO:3)and the heavy chain CDR sequences CDR1 (SYWLH, SEQ ID NO:4), CDR2(YINPRNDYTEYNQNFKD, SEQ ID NO:5), and CDR3 (RDITTFY, SEQ ID NO:6). 32.The method of claim 29, wherein the anti-CD22 antibody or fragmentthereof is selected from the group consisting of epratuzumab, 1F5,HIB22, 1-PC1, LT22, MEM-1, R1-B4, bu59, fpc1, mc64-12 and IS7.
 33. Themethod of claim 29, further comprising administering to said individualan anti-B cell antibody or antigen-binding fragment thereof that bindsto an antigen selected from the group consisting of CD5, CD15, CD19,CD20, CD21, CD22, CD23, CD30, CD33, CD37, CD38, CD40, CD40L, CD46, CD52,CD74, CD79a, CD80, CD138, HLA-DR and VEGF.
 34. The method of claim 29,wherein the immunomodulator is selected from the group consisting ofcytokines, lymphokines, monokines, stem cell growth factors,lymphotoxins, hematopoietic factors, colony stimulating factors (CSF),interferons (IFN), parathyroid hormone, thyroxine, insulin, proinsulin,relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroidstimulating hormone (TSH), luteinizing hormone (LH), hepatic growthfactor, prostaglandin, fibroblast growth factor, prolactin, placentallactogen, OB protein, transforming growth factor (TGF), TGF-α, TGF-β,insulin-like growth factor (IGF), erythropoietin, thrombopoietin, tumornecrosis factor (TNF), TNF-α, TNF-β, mullerian-inhibiting substance,mouse gonadotropin-associated peptide, inhibin, activin, vascularendothelial growth factor, integrin, interleukin (IL),granulocyte-colony stimulating factor (G-CSF), granulocytemacrophage-colony stimulating factor (GM-CSF), interferon-α,interferon-β, interferon-γ, S1 factor, IL-1, IL-1α, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18 IL-21, IL-23, IL-25, LIF, kit-ligand, FLT-3,angiostatin, thrombospondin and endostatin.
 35. The method of claim 29,wherein the immunomodulator is interferon-α.
 36. The method of claim 29,wherein the drug is selected from the group consisting of aplidin,azaribine, anastrozole, azacytidine, bleomycin, bortezomib,bryostatin-1, busulfan, calicheamycin, camptothecin,10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin,irinotecan (CPT-11), SN-38, carboplatin, cladribine, cyclophosphamide,cytarabine, dacarbazine, docetaxel, dactinomycin, daunomycinglucuronide, daunorubicin, dexamethasone, diethylstilbestrol,doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, ethinylestradiol, estramustine, etoposide, etoposide glucuronide, etoposidephosphate, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO),fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine,hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide,L-asparaginase, leucovorin, lomustine, mechlorethamine,medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine,6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin,mitotane, phenyl butyrate, prednisone, procarbazine, paclitaxel,pentostatin, PSI-341, semustine streptozocin, tamoxifen, taxanes, taxol,testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, velcade, vinblastine, vinorelbine andvincristine.
 37. The method of claim 29, wherein the drug is SN-38. 38.The method of claim 29, wherein the anti-angiogenic agent is selectedfrom the group consisting of angiostatin, endostatin, baculostatin,canstatin, maspin, an anti-VEGF binding molecule, an anti-placentalgrowth factor binding molecule and an anti-vascular growth factorbinding molecule.
 39. The method of claim 29, wherein the autoimmunedisease is selected from the group consisting of acute immunethrombocytopenia, chronic immune thrombocytopenia, dermatomyositis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, polyglandular syndromes, bullouspemphigoid, pemphigus vulgaris, diabetes mellitus (e.g., juvenilediabetes), Henoch-Schonlein purpura, post-streptococcal nephritis,erythema nodosum, Takayasu's arteritis, Addison's disease, rheumatoidarthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, perniciousanemia, rapidly progressive glomerulonephritis, psoriasis and fibrosingalveolitis.
 40. The method of claim 29, wherein the disease is systemiclupus erythematosus (SLE), Sjogren's syndrome or rheumatoid arthritis.41. The method of claim 29, further comprising killing B cells by amechanism selected from the group consisting of homotypic adhesion, lossof mitochondrial membrane potential, production of reactive oxygenspecies, increased phosphorylation of ERKs and JNK, downregulation ofpAkt and Bcl-xL, and enlargement of lysosomes.
 42. The method of claim29, wherein the anti-CD22 antibody is a G1m3 allotype.
 43. The method ofclaim 29, wherein the anti-CD22 antibody is a chimeric, humanized orhuman antibody.
 44. The method of claim 29, wherein the anti-CD22antibody or fragment thereof comprises human IgG1, IgG2, IgG3, or IgG4constant regions.